Antimicrobial peptides (AMPs) are promising novel antibiotics since they have shown antimicrobial activity against a wide range of bacterial species, including multiresistant bacteria; however, ...toxicity is the major barrier to convert antimicrobial peptides into active drugs. A profound and proper understanding of the complex interactions between these peptides and biological membranes using biophysical tools and model membranes seems to be a key factor in the race to develop a suitable antimicrobial peptide therapy for clinical use. In the search for such therapy, different combined approaches with conventional antibiotics have been evaluated in recent years and demonstrated to improve the therapeutic potential of AMPs. Some of these approaches have revealed promising additive or synergistic activity between AMPs and chemical antibiotics. This review will give an insight into the possibilities that physicochemical tools can give in the AMPs research and also address the state of the art on the current promising combined therapies between AMPs and conventional antibiotics, which appear to be a plausible future opportunity for AMPs treatment.
This review summarizes the theory of zeta potential (ZP) and the most relevant data about how it has been used for studying bacteria. We have especially focused on the discovery and characterization ...of novel antimicrobial compounds. The ZP technique may be considered an indirect tool to estimate the surface potential of bacteria, a physical characteristic that is key to maintaining optimal cell function. For this reason, targeting the bacterial surface is of paramount interest in the development of new antimicrobials. Surface-acting agents have been found to display a remarkable bactericidal effect and have simultaneously revealed a low tendency to trigger resistance. Changes in the bacterial surface as a result of various processes can also be followed by ZP measurements. However, due to the complexity of the bacterial surface, some considerations regarding the assessment of ZP must first be taken into account. Evidence on the application of ZP measurements to the characterization of bacteria and biofilm formation is presented next. We finally discuss the feasibility of using the ZP technique to assess antimicrobial-induced changes in the bacterial surface. Among these changes are those related to the interaction of the agent with different components of the cell envelope, membrane permeabilization, and loss of viability.
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•Advanced instruments to measure the ZP in an easy and economic way underline the applications of this technique to bacteria.•Zeta potential can be used as a diagnostic tool in bacteria.•Zeta potential can provide information about membrane permeability and viability, among other variables, in bacteria.•Zeta potential has an enormous potential for assessing the interactions of novel antimicrobial compounds with bacteria.
In the search for new antimicrobial molecules, antimicrobial peptides (AMPs) offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to ...membrane disruption and eventually cell death. In particular, the group of linear α-helical cationic peptides has attracted increasing research and clinical interest. The AMP P5 has been previously designed as a cationic linear α-helical sequence, being its antimicrobial and hemolytic properties also evaluated. In this work, we analyzed the feasibility of using P5 against a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa, one of the most common and risky pathogens in clinical practice. After antimicrobial activity confirmation in in vitro studies, synergistic activity of P5 with meropenem was evaluated, showing that P5 displayed significant synergistic activity in a time kill curve assay. The ability of P5 to permeabilize the outer membrane of P. aeruginosa can explain the obtained results. Finally, the antibiofilm activity was investigated by viability analysis (MTT assay), crystal violet and confocal imaging, with P5 displaying mild biofilm inhibition in the range of concentrations tested. Regarding biofilm disruption activity, P5 showed a higher efficacy, interfering with biofilm structure and promoting bacterial cell death. Atomic force microscope images further demonstrated the peptide potential in P. aeruginosa biofilm eradication, confirming the promising application of P5 in multi-resistant infections therapeutics.
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•The antimicrobial peptide P5 displays synergistic activity with meropenem in a carbapenem-resistant strain of P. aeruginosa.•P5 permeablizes the membrane of P. aeruginosa.•P5 eradicates P.aeruginosa biofilms and displays bactericidal activity against biofilm associated bacteria.
In the last years, the decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. This situation has brought ...attention to other antimicrobial agents like antimicrobial peptides (AMPs), for being considered an alternative to conventional drugs. These compounds target bacterial membranes for their activity, which gives them a broad spectrum of action and less probable resistance development. That is why the peptide-membrane interaction is a crucial aspect to consider in the study of AMPs. The aim of this work was the characterization of the “de novo” designed peptide P1, studying its interactions with model membranes (i.e. liposomes of DMPC:DMPG 5:1) in order to evaluate the final position of the peptide upon interacting with the membrane. Also, we tested the effects of the peptide in gram-positive and gram-negative bacteria. Later, by spectroscopic methods, the ability of the peptide to permeabilize the inner and outer membrane of E. coli and plasmatic membrane of S. aureus was assessed. The results obtained confirmed that P1 can disrupt both membranes, showing some difference in its activity as a function of the nature of each bacterial cell wall, confirming higher effects on gram-positive S. aureus. Finally, we also showed the ability of P1 to inhibit biofilms of that gram-positive bacterium.
All data obtained in this work allowed us to propose a model, where the first interactions of the peptide with the bacterial envelope, seem to depend on the gram-negative and gram-positive cell wall structure. After that first interaction, the peptide is stabilized by Trp residues depth inserted into the hydrocarbon region, promoting several changes in the organization of the lipid bilayer, following a carpet-like mechanism, which results in permeabilization of the membrane, triggering the antimicrobial activity.
•The difference in gram-negative and gram-positive envelop play a key role in the interaction of P1.•P1 acts through the carpet-like model, covering the surface in parallel alignments to the membrane.•Cooperativity and kinetics of membrane permeabilization are key factors in antimicrobial activity.•P1 was able to inhibit the biofilm formation of S. aureus at sub-MIC concentrations.
Antimicrobial peptides are small molecules that display antimicrobial activity against a wide range of pathogens. In a previous work, by using model membranes we studied P6, a peptide that shows no ...antimicrobial activity, and P6.2, which exhibits antibacterial activity. In the present work we aimed to unravel the mode of action of these peptides by studying their interaction in vivo with Escherichia coli and Staphylococcus aureus. In this sense, to study the interactions with bacterial cells and their effect on the bacterial surface, zeta potential, spectroscopic, and microscopic methodologies were applied. P6.2 exhibits a higher affinity toward both bacterial envelopes. The ability of both peptides to disrupt afterwards the bacterial membrane was also studied. Both peptides were able to induce bacterial membrane damage, but higher concentrations of P6 were needed to obtain results comparable to those obtained for P6.2. Additionally, P6.2 exhibited faster damage kinetics. Altogether, these data allow postulating, in a physiologic model, that the lower affinity of P6 for bacterial envelope results in a minor final concentration of the peptide in the bacterial membrane unable to trigger the antimicrobial activity. Finally, the fact that the active P6.2 has the same MIC value for the Gram-positive and Gram-negative bacteria tested, but not the same profile in the permeabilization assays, reinforces the question of whether cell wall components act as electrostatic barriers preventing or minimizing membrane-active AMPs lethal action at the membrane level.
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•Achieving the permeabilization of the inner membrane seems to be the critical step in the P6.2 killing mechanism in E. coli.•In S. aureus faster full permeabilization takes place probably as they lack outer membrane.•Fast membrane disruption kinetics is a key feature to avoid bacteria to overcome the damage.•AFM data revealed that P6.2 induces leakage of bacterial content and bubbles on the bacterial membrane.
Colistin is a polymyxin antibiotic (polymyxin E) that has in recent years re-emerged as an option for treatment of multidrug-resistant bacteria. Recently, the re-introduction of colistin resulted in ...the appearance of colistin-resistant bacteria, which is usually caused by LPS modifications. The fact that this modification is mediated by a plasmid carrying the mcr-1 gene, implies a horizontal transfer of colistin resistance. In Argentina, the National Reference Laboratory in Antimicrobial Resistance (NRLAR), has recently screened several bacteria for the MCR-1 plasmid, detecting nine
Escherichia coli
isolates carrying the plasmid with the mcr-1 gene, among others. In this context, we proposed to assess the effect of surface charge modifications induced by the plasmid MCR-1 and its impact on the resulting colistin resistance in two clinical isolates of colistin-resistant
E. coli
. Using zeta potential assays, we confirmed the reduction of negative charge exposure on clinical isolates compared to the reference strain of
E. coli
. In addition, through permeabilization assays, we were able to correlate this reduction in charge exposure with the extent of damage to the bacterial membrane. The fact that this surface charge modification through substitution of lipid A is plasmid encoded, represents an important concern for future antimicrobial peptide drug development.
Antimicrobial peptides (AMPs) are one of the elements of innate immunity that have a crucial role in fighting infections. These molecules are produced by all kinds of cells and can display a wide ...spectrum of action against bacterial or fungal infections. Bacterial resistance toward broad‐spectrum antibiotics has become a major concern in recent years. In this context, AMPs have emerged as promising alternative therapy in response to this increasing problem because of their ability to inhibit growth and biofilm formation, even in drug‐resistant microorganisms. However, despite the myriad of patents filed related to AMPs, only a small proportion of AMPs have already received certification, either by the Food and Drug Administration (FDA) or European Medicines Agency (EMA). In order to pave the way of AMPs to certification, a comprehensive knowledge of the mechanism of action of these peptides is essential. Several models have been proposed to explain it and the permeabilization of the bacterial envelope seems to play a key role in most of them. In this review, we describe the different techniques that are generally used to assess the permeabilization induced by AMPs in either in vivo or in vitro systems. Additionally, a comprehensive analysis of the cooperation during the process of binding and permeabilization is included.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O processamento de imagens é uma área promissora na automação, por poder ser aplicado nas mais variadas atividades da tecnologia como, por exemplo, na medicina, na agricultura de precisão, dentre muitas outras. Este trabalho consiste na aplicação da área de processamento de imagens, voltada a área de segmentação, com os operadores de bordas Roberts, Prewitt e Sobel. Tendo também muita contribuição na área de processamento de imagens e sistemas embarcados, implementando os detectores de bordas na placa FPGA (Field Programmable Gate. Array), por meio de software e de simulações do hardware. A configuração do processador NIOS permitiu a instalação do sistema operacional uClinux e de um software descrito na linguagem C ANSI com a imagem em níveis de cinza particionada em quatro sub- imagens. O hardware gerado, foi modelado com a linguagem de descrição de hardware VHDL (VHSIC – Hardware Description Language). Para serem comparadas às imagens geradas, detectores de bordas no ambiente MATLAB foram aplicado por ser uma ferramenta conhecida, usual, com funções para aplicações na área de processamento de imagens. Para ter um melhor entendimento sobre os algoritmos de detecção de bordas, foram criados algoritmos na linguagem C ANSI
Image processing is a promising area for automation, because it can be applied in a variety of technology activities, for example, in medicine, precision agriculture, among many others. This work is the application of image processing area, facing the segmentation area, with the operators of edges Roberts, Prewitt and Sobel. Also having a lot of contribution in the field of image processing and embedded systems, implementing the edge detectors in the FPGA board by means of simulation software and hardware. The NIOS processor configuration allowed the installation of the uClinux operating system and software described in the ANSI C language with the image in grayscale partitioned into four sub-images. The hardware generated, was modeled with the hardware description language VHDL (VHSIC - Hardware Description Language). To be compared to the images generated, edge detectors were implemented in MATLAB, a tool known, usual, with functions for applications in image processing. To get a better understanding of the edge detection algorithms were created algorithms in ANSI C language
Mutations in
PINK1
(
PARK6
), a serine/threonine kinase involved in mitochondrial homeostasis, are associated with early onset Parkinson’s disease. Fibroblasts from Parkinson’s disease patients with ...compound heterozygous mutations in exon 7 (c.1488 + 1G > A; c.1252_1488del) showed no apparent signs of mitochondrial impairment. To elucidate changes primarily caused by lack of functional PINK1, we over-expressed wild-type
PINK1
, which induced a significant downregulation of
LRRK2
(
PARK8
). Indeed, we found that LRRK2 protein basal levels were significantly higher in the mutant
PINK1
fibroblasts. To examine the interaction between the two
PARK
genes in a disease-relevant cell context, we generated induced pluripotent stem cell (iPSC) lines from mutant, carrier and control fibroblasts by lentiviral-mediated re-programming. Efficiency of neural induction and dopamine differentiation using a floor-plate induction protocol was similar in all genotypes. As observed in fibroblasts,
PINK1
mutant neurons showed increased LRRK2 expression both at the RNA and protein level and transient over-expression of wild-type
PINK1
efficiently downregulated
LRRK2
levels. Additionally, we confirmed a dysregulation of
LRRK2
expression in fibroblasts from patients with a different homozygous mutation in
PINK1
exon 4, c.926G > A (G309D). Thus, our results identify a novel role of PINK1 modulating the levels of LRRK2 in Parkinson’s disease fibroblasts and neurons, suggest a convergent pathway for these
PARK
genes, and broaden the role of LRRK2 in the pathogenesis of Parkinson’s disease.
Fibronectin fibrillogenesis is the physiological process by which cells elaborate a fibrous FN matrix. Poly(ethyl acrylate), PEA, has been described to induce a similar process upon simple ...adsorption of fibronectin (FN) from a protein solutionin the absence of cellsleading to the so-called material-driven fibronectin fibrillogenesis. Poly(methyl acrylate), PMA, is a polymer with very similar chemistry to PEA, on which FN is adsorbed, keeping the globular conformation of the protein in solution. We have used radical polymerization to synthesize copolymers with controlled EA/MA ratio, seeking to modulate the degree of FN fibrillogenesis. The physicochemical properties of the system were studied using dynamic–mechanical analysis, differential scanning calorimetry, and water contact angle. Both the degree of FN fibrillogenesis and the availability of the integrin binding region of FN directly depend on the percentage of EA in the copolymer, whereas the same total amount of FN was adsorbed regardless the EA/MA ratio. Cell morphology adhesion and differentiation of murine C2C12 were shown to depend on the degree of FN fibrillogenesis previously attained on the material surface. Myogenic differentiation was enhanced on the copolymers with higher EA content, i.e. more interconnected FN fibrils.
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