Antibacterial activity of a novel antimicrobial peptide [W7]KR12-KAEK derived from KR-12 against Streptococcus mutans planktonic cells and biofilms.

The aims of this study were to describe the synthesis of a novel synthetic peptide based on the primary structure of the KR-12 peptide and to evaluate its antimicrobial and anti-biofilm activities against Streptococcus mutans. The antimicrobial effect of KR-12 and [W7]KR12-KAEK was assessed by determining the minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations. The evaluation of anti-biofilm activity was assessed through total biomass quantification, colony forming unit counting and scanning electron microscopy. [W7]KR12-KAEK showed MIC and MBC values ranging from 31.25 to 7.8 and 62.5 to 15.6 µg ml-1, respectively. Furthermore, [W7]KR12-KAEK significantly reduced biofilm biomass (50-100%). Regarding cell viability, [W7]KR12-KAEK showed reductions in the number of CFUs at concentrations ranging from 62.5 to 7.8 µg ml-1 and 500 to 62.5 µg ml-1 with respect to biofilm formation and preformed biofilms, respectively. SEM micrographs of S. mutans treated with [W7]KR12-KAEK suggested damage to the bacterial surface. [W7]KR12-KAEK is demonstrated to be an antimicrobial agent to control microbial biofilms.

Effect of dimerization on the mechanism of action of aurein 1.2.

The mechanism of action of antimicrobial peptides depends on physicochemical properties such as structure, concentration, and oligomerization. Here, we focused on the effect of dimerization on the mechanism of action of aurein 1.2 (AU). We designed a lysine-linked AU dimer, (AU)2K, and its interaction with membrane mimetics was studied using four biophysical techniques and molecular dynamics simulations. Circular dichroism and molecular dynamics studies showed that AU displayed a typical spectrum for disordered structures in aqueous solution whereas (AU)2K exhibited the typical spectrum of α-helices in a coiled-coil conformation, wherein helices are wrapped around each other. With the addition of large unilamellar vesicles (LUVs), AU adopted an α-helix structure whereas the coiled-coil structure of (AU)2K assumed an extended conformation. Carboxyfluorescein release experiments with LUVs showed that both peptides were able to permeabilize vesicles although the leakage response to increases in peptide concentration differed. Optical microscopy experiments showed that both peptides induced pore opening and the dimer eventually caused the vesicles to burst. Finally, calorimetric traces determined by isothermal titration calorimetry on the LUVs also showed significant differences in peptide-membrane interactions. Together, the results of our study demonstrated that dimerization changes the mechanism of action of AU.

Dimerization of aurein 1.2: effects in structure, antimicrobial activity and aggregation of Cândida albicans cells.

Antimicrobial peptides (AMPs) are a promising solution to face the antibiotic-resistant problem because they display little or no resistance effects. Dimeric analogues of select AMPs have shown pharmacotechnical advantages, making these molecules promising candidates for the development of novel antibiotic agents. Here, we evaluate the effects of dimerization on the structure and biological activity of the AMP aurein 1.2 (AU). AU and the C- and N-terminal dimers, (AU)2K and E(AU)2, respectively, were synthesized by solid-phase peptide synthesis. Circular dichroism spectra indicated that E(AU)2 has a "coiled coil" structure in water while (AU)2K has an α-helix structure. In contrast, AU displayed typical spectra for disordered structures. In LPC micelles, all peptides acquired a high amount of α-helix structure. Hemolytic and vesicle permeabilization assays showed that AU has a concentration dependence activity, while this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action of AU. Notably, the antimicrobial activity against bacteria and yeast decreased with dimerization. However, dimeric peptides promoted the aggregation of C. albicans. The ability to aggregate yeast cells makes dimeric versions of AU attractive candidates to inhibit the adhesion of C. albicans to biological targets and medical devices, preventing disease caused by this fungus.

Effects of dimerization on the structure and biological activity of antimicrobial peptide Ctx-Ha.

It is well known that cationic antimicrobial peptides (cAMPs) are potential microbicidal agents for the increasing problem of antimicrobial resistance. However, the physicochemical properties of each peptide need to be optimized for clinical use. To evaluate the effects of dimerization on the structure and biological activity of the antimicrobial peptide Ctx-Ha, we have synthesized the monomeric and three dimeric (Lys-branched) forms of the Ctx-Ha peptide by solid-phase peptide synthesis using a combination of 9-fluorenylmethyloxycarbonyl (Fmoc) and t-butoxycarbonyl (Boc) chemical approaches. The antimicrobial activity assay showed that dimerization decreases the ability of the peptide to inhibit growth of bacteria or fungi; however, the dimeric analogs displayed a higher level of bactericidal activity. In addition, a dramatic increase (50 times) in hemolytic activity was achieved with these analogs. Permeabilization studies showed that the rate of carboxyfluorescein release was higher for the dimeric peptides than for the monomeric peptide, especially in vesicles that contained sphingomyelin. Despite different biological activities, the secondary structure and pore diameter were not significantly altered by dimerization. In contrast to the case for other dimeric cAMPs, we have shown that dimerization selectively decreases the antimicrobial activity of this peptide and increases the hemolytic activity. The results also show that the interaction between dimeric peptides and the cell wall could be responsible for the decrease of the antimicrobial activity of these peptides.

MULTIMERIN2 impairs tumor angiogenesis and growth by interfering with VEGF-A/VEGFR2 pathway.

MULTIMERIN2 (MMRN2), also known as Endoglyx-1, is an extracellular matrix glycoprotein whose function has so far remained elusive. Given its specific localization in tight association with the endothelium we hypothesized that this protein could modulate neo-angiogenesis. By multiple assays we showed that MMRN2 significantly impaired endothelial cell (EC) migration and organization of a functional vessel network. The interaction of ECs with MMRN2 induced a striking impairment of VEGFR1 and VEGFR2 activation. We focused our attention on VEGFR2, a chief regulator of angiogenesis, and clarified that MMRN2 interfered with the VEGF/VEGFR2 axis through a direct binding with VEGF-A. This novel interaction was assessed in several assays and the affinity was estimated (Kd ∼50 nM). We next questioned whether the anti-angiogenic properties of MMRN2 could impair tumor growth. Although overexpression of MMRN2 by HT1080 cells did not affect their growth and apoptotic rate in vitro, it remarkably affected their growth in vivo. In fact, MMRN2-positive cells failed to efficiently grow and form well-vascularized tumors; a similar outcome was observed following treatment of established tumors with a MMRN2 adenoviral construct. Tumor-section immunostaining revealed a strong co-localization of VEGF-A with the ectopically expressed MMRN2. These novel findings suggest that VEGF may be sequestered by MMRN2 and be less available for the engagement to the receptors. Taken together these results highlight MMRN2 as a crucial player in the regulation of EC function, neo-angiogenesis and hence tumor growth. We hypothesize that secreted and deposited MMRN2 may function as a homeostatic barrier halting the sprouting of novel vessels, and suggest that these studies may embody the potential for the development of novel tools for cancer treatment.

Extracellular matrix: a matter of life and death.

Extracellular matrix (ECM) is an essential component of the stromal microenvironment both from a structural and a functional point of view. The ECM functions as a scaffold for tissue organization and regulates growth factors and chemokines availability thus contributing to maintain tissue homeostasis. Attachment of cells to ECM is essential to support cell survival, growth, and proliferation, and the lack of these interactions can trigger a type of cell death named anoikis. Several studies point out that alterations of ECM composition are often responsible of many pathological conditions such as cancer, of which it has been demonstrated to be occasionally the main promoter. ECM does not always represent a prosurvival stimulus; among the different array of ECM molecules a set of proteins can negatively affect cell viability and are thought to play an important role in tumor progression. For this reason attention has been focused on these molecules as potential tools or targets for therapy.