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Projeto de investigação
Towards the development of antimicrobial peptides active against bacterial biofilms
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Structural determinants conferring unusual long life in human serum to rattlesnake‐derived antimicrobial peptide Ctn(15‐34)
Publication . Pérez‐Peinado, Clara; Dias, Susana; Mendonça, Diogo A.; Castanho, Miguel A. R. B.; Veiga, Ana Salomé; Andreu, David
Ctn[15-34], a downsized version of the snake venom cathelicidin-like peptide crotalicidin (Ctn), shows an unusually high lifespan (t1/2 , approximately 12 h) in human serum, which significantly adds to its promise as an antimicrobial and antitumor agent. Herein we investigate the role of Ctn[15-34] structure on serum survival. Using a set of analogs, we show that C-terminal amidation, as well as the specific layout of the Ctn[15-34] sequence-a helical N-terminal domain followed by a hydrophobic domain-is crucial for slow degradation, and any change in their arrangement results in significantly lower t1/2 . Aside from the privileged primary structure, features such as self-aggregation can be ruled out as causes for the long serum life. Instead, studies in other protease-rich fluids suggest a key role for certain human serum components. Finally, we demonstrate that Ctn[15-34] is able to induce bacterial death even after 12-hour pre-incubation in serum, in agreement with the proteolytic data. Altogether, the results shed light on the uncommon stability of Ctn[15-34] in human serum and confirm its potential as an anti-infective lead.
Synthesis and characterization of peptide-chitosan conjugates (PepChis) with lipid bilayer affinity and antibacterial activity
Publication . Costa Petrin, Thais H.; Fadel, Valmir; Martins, Danubia B.; Dias, Susana; Cruz, Ana; Sergio, Luciana Marciano; Arcisio-Miranda, Manoel; Castanho, Miguel A. R. B.; Cabrera, Marcia P. dos Santos
Antimicrobial peptides appear among innovative biopolymers with potential therapeutic interest. Nevertheless, issues concerning efficiency, production costs, and toxicity persist. Herein, we show that conjugation of peptides with chitosans can represent an alternative in the search for these needs. To increase solubility, deacetylated and degraded chitosans were prepared. Then, they were functionalized via N-succinimidyl-S-acetylthiopropionate or via glutathione (GSH), an endogenous peptide linker. To the best of our knowledge, it is the first time that GSH is used as a thiolating agent for the conjugation of peptides. Next, thiolated chitosans were conjugated through a disulfide bond with designed shortchain peptides, one of them derived from the antimicrobial peptide Jelleine-I. Conjugates and respective reaction intermediates were characterized by absorciometry, attenuated total reflectance−Fourier transform infrared, and 1H NMR. Zeta potential measurements showed the cationic nature of these biomacromolecules and their preferential partitioning to Gram-positive bacterial-like model membranes. In vitro investigation using representative Gram-positive and -negative bacteria (Staphylococcus aureus and Escherichia coli, respectively) showed that the conjugation strategies lead to enhanced activity in relation to the unconjugated peptide and to the unconjugated chitosan. The obtained products showed selectivity toward S. aureus at low cytotoxicity as determined in NIH/3T3 cells. Overall, our study demonstrates that an appropriate choice of antimicrobial peptide and chitosan characteristics leads to increased antimicrobial activity of the conjugated product and represents a strategy to modulate the activity and selectivity of antimicrobials resorting to low-cost chemicals. The present proposal starts from less expensive raw materials (chitosan and short-chain peptide), is based on aqueous solvents, and minimizes the use of reactants with a higher environmental impact. The final biopolymer contains the backbone of chitosan, just 3−6% peptide derived from royal jelly and GSH, all of them considered safe for human use or as a physiological molecule.
The mechanism of action of pepR, a viral-derived peptide, against Staphylococcus aureus biofilms
Publication . Pinto, Sandra; Dias, Susana; Cruz, Ana F; Mil-Homens, Dalila; Fernandes, Fabio; Valle, Javier; Andreu, David; Prieto, Manuel; Castanho, Miguel A. R. B.; Coutinho, Ana; Veiga, Ana Salomé
Objectives: To investigate the mechanism of action at the molecular level of pepR, a multifunctional peptide derived from the Dengue virus capsid protein, against Staphylococcus aureus biofilms.
Methods: Biofilm mass, metabolic activity and viability were quantified using conventional microbiology techniques, while fluorescence imaging methods, including a real-time calcein release assay, were employed to investigate the kinetics of pepR activity at different biofilm depths.
Results: Using flow cytometry-based assays, we showed that pepR is able to prevent staphylococcal biofilm formation due to a fast killing of planktonic bacteria, which in turn resulted from a peptide-induced increase in the permeability of the bacterial membranes. The activity of pepR against pre-formed biofilms was evaluated through the application of a quantitative live/dead confocal laser scanning microscopy (CLSM) assay. The results show that the bactericidal activity of pepR on pre-formed biofilms is dose and depth dependent. A CLSM-based assay of calcein release from biofilm-embedded bacteria was further developed to indirectly assess the diffusion and membrane permeabilization properties of pepR throughout the biofilm. A slower diffusion and delayed activity of the peptide at deeper layers of the biofilm were quantified.
Conclusions: Overall, our results show that the activity of pepR on pre-formed biofilms is controlled by its diffusion along the biofilm layers, an effect that can be counteracted by an additional administration of peptide. Our study sheds new light on the antibiofilm mechanism of action of antimicrobial peptides, particularly the importance of their diffusion properties through the biofilm matrix on their activity.
Towards the development of antimicrobial peptides active against bacterial biofilms
Publication . Dias, Susana Filipa Almeida; Veiga, Ana Salomé Rocha do Nascimento
Antibiotic-resistant bacteria are rapidly emerging, and the number of available therapeutic options to fight them is decreasing. In addition, as a surviving strategy, bacteria can aggregate and form biofilms, a multicellular community embedded in a self-produced matrix of extracellular polymeric substances (EPS), which contributes significantly to the reduced susceptibility of bacteria towards conventional antibiotics and innate host defenses. It has been estimated that biofilms are responsible for most infections in tissues and are also recurrent colonizers of biomedical devices. Hence, the identification of novel antimicrobial agents capable to fight infections caused by resistant bacteria and bacterial biofilms is urgently needed. Antimicrobial peptides (AMPs) have been proposed as promising therapeutic alternatives to conventional antibiotics due to their broad-spectrum activity, fast-killing kinetics, and distinct mechanisms of action. In general, AMPs selectively target and kill bacteria through membrane-disruptive mechanisms, thus their activity is independent of the cellular metabolism. This feature confers AMPs the ability to act on dormant populations, which are frequently found in biofilms and are particularly difficult to target by conventional antibiotics. Taking advantage of the fact that viral proteins are an underexplored source of bioactive peptides with antimicrobial properties and based on the knowledge that cell-penetrating peptides (CPPs) can act as AMPs, the present work identified one viral-derived peptide, vCPP2319, active against bacteria in the planktonic and biofilm forms. The peptide was able to effectively kill bacteria through a mechanism of action involving bacterial membrane permeabilization. Nonetheless, vCPP2319 had a limited effect on the biofilm EPS matrix itself. Thus, biofilm treatment with vCPP2319 and a matrix-degrading enzyme, α-amylase, was studied. This combination did not improve the antibacterial action of the peptide. Given the knowledge that cyclization of bioactive peptides is a promising approach for improving peptides’ stability and bioactivity, the antibiofilm activity of the cyclic peptide, [G1K,K8R]cGm, was investigated against S. aureus biofilms. The peptide was able to kill biofilm-embedded cells in a concentration-dependent manner. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes with a half time of 65 min. Also, we tested an analogue of [G1K,K8R]cGm without disulfide bonds, and a linear unfolded analogue, and found both to be inactive. This finding suggests that the three dimensional structure of [G1K,K8R]cGm and its stabilization by disulfide bonds are essential for its antibacterial and antibiofilm activities. Overall, this study demonstrates the potential of viral proteins as rich sources of new bioactive peptides with antibacterial and antibiofilm properties. In addition, peptide backbone cyclization proved to be valuable strategy to enhance AMPs activity against preformed biofilms.
A designed cyclic analogue of gomesin has potent activity against Staphylococcus aureus biofilms
Publication . Dias, Susana; Pinto, Sandra; Silva-Herdade, Ana S.; Cheneval, Olivier; Craik, David J; Coutinho, Ana; Castanho, Miguel A. R. B.; Henriques, Sónia T.; Veiga, Ana Salomé
Background: Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed.
Objectives: To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections.
Methods: We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide.
Results: The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive.
Conclusions: The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.
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Fundação para a Ciência e a Tecnologia
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PD/BD/114425/2016