Structural and Mechanistic Evidence for Calcium Interacting Sites in the HIV Transmembrane Protein gp41 Involved in Membrane Fusion.

The HIV envelope protein gp160 comprises two subunits, gp120 and gp41, responsible for receptor binding and membrane fusion during viral entry, respectively. In the course of the membrane fusion process, gp41 undergoes a conformational change, leading to the formation of a six-helix bundle (SHB), which ultimately drives membrane fusion. The gp41 C-terminal and N-terminal heptad repeats (CHR and NHR) interact with one another to form the SHB, and this step can be targeted by peptide inhibitors, which are used in the clinic to mitigate HIV infection. Here, we discover the calcium interaction motifs (CIMs) in the gp41 CHR and NHR regions via NMR spectroscopy. We find that the assembly of the CHR-NHR SHB is facilitated in Ca2+-containing media and impaired in CIM mutants. Of note, the clinically approved, gp41-derived fusion inhibitor T20, which does not contain the CIM motif, exhibits reduced inhibitory efficiency when challenged with calcium. This finding could have important implications for the development of better fusion inhibitors for HIV.

TLR2 Dimerization Blockade Allows Generation of Homeostatic Intestinal Macrophages under Acute Colitis Challenge.

Recruited blood monocytes contribute to the establishment, perpetuation, and resolution of tissue inflammation. Specifically, in the inflamed intestine, monocyte ablation was shown to ameliorate colitis scores in preclinical animal models. However, the majority of intestinal macrophages that seed the healthy gut are also monocyte derived. Monocyte ablation aimed to curb inflammation would therefore likely interfere with intestinal homeostasis. In this study, we used a TLR2 trans-membrane peptide that blocks TLR2 dimerization that is critical for TLR2/1 and TLR2/6 heterodimer signaling to blunt inflammation in a murine colitis model. We show that although the TLR2 peptide treatment ameliorated colitis, it allowed recruited monocytes to give rise to macrophages that lack the detrimental proinflammatory gene signature and reduced potentially damaging neutrophil infiltrates. Finally, we demonstrate TLR blocking activity of the peptide on in vitro cultured human monocyte-derived macrophages. Collectively, we provide a significantly improved anti-inflammatory TLR2 peptide and critical insights in its mechanism of action toward future potential use in the clinic.

Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides.

The increasing number of resistant bacteria is a major threat worldwide, leading to the search for new antibiotic agents. One of the leading strategies is the use of antimicrobial peptides (AMPs), cationic and hydrophobic innate immune defense peptides. A major target of AMPs is the bacterial membrane. Notably, accumulating data suggest that AMPs can activate the two-component systems (TCSs) of Gram-negative bacteria. These include PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB), responsible for remodeling of the bacterial cell surface. To better understand this mechanism, we utilized bacteria deficient either in one system alone or in both and biophysical tools including fluorescence spectroscopy, single-cell atomic force microscopy, electron microscopy, and mass spectrometry (Moskowitz, S. M.; Antimicrob. Agents Chemother. 2012, 56, 1019-1030; Cheng, H. Y.; J. Biomed. Sci. 2010, 17, 60). Our data suggested that the two systems have opposing effects on the properties of Salmonella enterica. The knockout of PhoPQ made the bacteria more susceptible to AMPs by making the surface less rigid, more polarized, and permeable with a slightly more negatively charged cell wall. In addition, the periplasmic space is thinner. In contrast, the knockout of PmrAB did not affect its susceptibility, while it made the bacterial outer layer very rigid, less polarized, and less permeable than the other two mutants, with a negatively charged cell wall similar to the WT. Overall, the data suggest that the coexistence of systems with opposing effects on the biophysical properties of the bacteria contribute to their membrane flexibility, which, on the one hand, is important to accommodate changing environments and, on the other hand, may inhibit the development of meaningful resistance to AMPs.

Fractal properties of cell surface structures: A view from AFM.

Cell surfaces are densely populated with various proteins. Aggregation of these proteins to nanoscale clusters can be critical for various cellular functions such as signaling, motility and division. Quantitative characterization of corresponding structures and their changes might be useful to understand these basic cell processes and serve as an early marker of cellular stress or diseases. Atomic force microscopy (AFM) allows high-resolution imaging of cell surface structures, resolving fine details of these structures. Moreover, AFM enables simultaneous imaging of cell surface morphology and mapping of its' mechanical characteristics. This review focuses on applying the fractal dimension measure as a sensitive method to quantify single cell surface structures and their changes from AFM images.

Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis.

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll-like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2-p) ameliorated DSS-induced colitis by interfering specifically with the activation of Ly6C(+) monocytes without affecting their recruitment to the colon. We report that TLR2-p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2-TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal-regulated kinases (ERK) signaling and reduced secretion of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-23, IL-12, and IL-1ß. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro-inflammatory Ly6C(hi) monocytes and suggests that inhibition of this aggregation by TLR2-p might have therapeutic potential in the treatment of acute gut inflammation.

The HTLV-1 gp21 fusion peptide inhibits antigen specific T-cell activation in-vitro and in mice.

The ability of the Lentivirus HIV-1 to inhibit T-cell activation by its gp41 fusion protein is well documented, yet limited data exists regarding other viral fusion proteins. HIV-1 utilizes membrane binding region of gp41 to inhibit T-cell receptor (TCR) complex activation. Here we examined whether this T-cell suppression strategy is unique to the HIV-1 gp41. We focused on T-cell modulation by the gp21 fusion peptide (FP) of the Human T-lymphotropic Virus 1 (HTLV-1), a Deltaretrovirus that like HIV infects CD4+ T-cells. Using mouse and human in-vitro T-cell models together with in-vivo T-cell hyper activation mouse model, we reveal that HTLV-1's FP inhibits T-cell activation and unlike the HIV FP, bypasses the TCR complex. HTLV FP inhibition induces a decrease in Th1 and an elevation in Th2 responses observed in mRNA, cytokine and transcription factor profiles. Administration of the HTLV FP in a T-cell hyper activation mouse model of multiple sclerosis alleviated symptoms and delayed disease onset. We further pinpointed the modulatory region within HTLV-1's FP to the same region previously identified as the HIV-1 FP active region, suggesting that through convergent evolution both viruses have obtained the ability to modulate T-cells using the same region of their fusion protein. Overall, our findings suggest that fusion protein based T-cell modulation may be a common viral trait.

The HIV gp41 Fusion Protein Inhibits T-Cell Activation through the Lentiviral Lytic Peptide 2 Motif.

The human immunodeficiency virus enters its host cells by membrane fusion, initiated by the gp41 subunit of its envelope protein. gp41 has also been shown to bind T-cell receptor (TCR) complex components, interfering with TCR signaling leading to reduced T-cell activation. This immunoinhibitory activity is suggested to occur during the membrane fusion process and is attributed to various membranotropic regions of the gp41 ectodomain and to the transmembrane domain. Although extensively studied, the cytosolic region of gp41, termed the cytoplasmic tail (CT), has not been examined in the context of immune suppression. Here we investigated whether the CT inhibits T-cell activation in different T-cell models by utilizing gp41-derived peptides and expressed full gp41 proteins. We found that a conserved region of the CT, termed lentiviral lytic peptide 2 (LLP2), specifically inhibits the activation of mouse, Jurkat, and human primary T-cells. This inhibition resulted in reduced T-cell proliferation, gene expression, cytokine secretion, and cell surface expression of CD69. Differential activation of the TCR signaling cascade revealed that CT-based immune suppression occurs downstream of the TCR complex. Moreover, LLP2 peptide treatment of Jurkat and primary human T-cells impaired Akt but not NFκB and ERK1/2 activation, suggesting that immune suppression occurs through the Akt pathway. These findings identify a novel gp41 T-cell suppressive element with a unique inhibitory mechanism that can take place post-membrane fusion.

Deficient Lipid A Remodeling by the arnB Gene Promotes Biofilm Formation in Antimicrobial Peptide Susceptible Pseudomonas aeruginosa.

Multidrug resistant bacteria possess various mechanisms that can sense environmental stresses such as antibiotics and antimicrobial peptides and rapidly respond to defend themselves. Two known defense strategies are biofilm formation and lipopolysaccharide (LPS) modification. Though LPS modifications are observed in biofilm-embedded bacteria, their effect on biofilm formation is unknown. Using biochemical and biophysical methods coupled with confocal microscopy, atomic force microscopy, and transmission electron microscopy, we show that biofilm formation is promoted in a Pseudomonas aeruginosa PAO1 strain with a loss of function mutation in the arnB gene. This loss of function prevents the addition of the positively charged sugar 4-amino-4-deoxy-l-arabinose to lipid A of LPS under restrictive magnesium conditions. The data reveal that the arnB mutant, which is susceptible to antimicrobial peptides, forms a biofilm that is more robust than that of the wild type. This is in line with the observations that the arnB mutant exhibits outer surface properties such as hydrophobicity and net negative charge that promote the formation of biofilms. Moreover, when grown under Mg2+ limitation, both the wild type and the arnB mutant exhibited a reduction in the level of membrane-bound polysaccharides. The data suggest that the loss of polysaccharides exposes the membrane and alters its biophysical properties, which in turn leads to more biofilm formation. In summary, we show for the first time that blocking a specific lipid A modification promotes biofilm formation, suggesting a trade-off between LPS remodeling and resistance mechanisms of biofilm formation.

Intramembrane attenuation of the TLR4-TLR6 dimer impairs receptor assembly and reduces microglia-mediated neurodegeneration.

Recently, a single study revealed a new complex composed of Toll-like receptor 4 (TLR4), TLR6, and CD36 induced by fibrillary Aß peptides, the hallmark of Alzheimer's disease. Unlike TLRs located on the plasma membrane that dimerize on the membrane after ligand binding to their extracellular domain, the TLR4-TLR6-CD36 complex assembly has been suggested to be induced by intracellular signals from CD36, similar to integrin inside-out signaling. However, the assembly site of TLR4-TLR6-CD36 and the domains participating in Aß-induced signaling is still unknown. By interfering with TLR4-TLR6 dimerization using a TLR4-derived peptide, we show that receptor assembly is abrogated within the plasma membrane. Furthermore, we reveal that the transmembrane domains of TLR4 and TLR6 have an essential role in receptor dimerization and activation. Inhibition of TLR4-TLR6 assembly was associated with reduced secretion of proinflammatory mediators from microglia cells, ultimately rescuing neurons from death. Our findings support TLR4-TLR6 dimerization induced by Aß. Moreover, we shed new light on TLR4-TLR6 assembly and localization and show the potential of inhibiting TLR4-TLR6 dimerization as a treatment of Alzheimer's disease.

HIV-1 fusion protein exerts complex immunosuppressive effects.

One of the routes by which HIV-1 is able to escape the immune response is by immunosuppression. The gp41 fusion protein of the HIV-1 envelope mediates virus entry by membrane fusion and also functions as an inhibitor of T cell activation. Here, we review the recent studies suggesting that some of the gp41 immunosuppressive processes are initiated by novel motifs, located within the hydrophobic regions of the protein. This indicates that the immunosuppressive process mediated by gp41 is much more complex than initially thought. Additionally, we propose a model illustrating the interactions and interferences of these regions with the T cell receptor complex.

Heat Shock Protein 90 Facilitates Latent HIV Reactivation through Maintaining the Function of Positive Transcriptional Elongation Factor b (p-TEFb) under Proteasome Inhibition.

The persistence of HIV in resting memory CD4+ T cells at a latent state is considered as the major barrier on the path to achieve a cure for HIV. Proteasome inhibitors (PIs) were previously reported as latency reversing agents (LRAs) but the mechanism underlying this function is yet unclear. Here we demonstrate that PIs reactivate latent HIV ex vivo without global T cell activation, and may facilitate host innate immune responses. Mechanistically, latent HIV reactivation induced by PIs is mediated by heat shock factor 1 (HSF1) via the recruitment of the heat shock protein (HSP) 90-positive transcriptional elongation factor b (p-TEFb) complex. Specifically, HSP90 downstream HSF1 gives positive feedback to the reactivation process through binding to cyclin-dependent kinase 9 (CDK9) and preventing it from undergoing degradation by the proteasome. Overall, these findings suggest proteasome inhibitors as potential latency reversing agents. In addition, HSF1/HSP90 involved in HIV transcription elongation, may serve as therapeutic targets in HIV eradication.

NMR structure and binding of esculentin-1a (1-21)NH2 and its diastereomer to lipopolysaccharide: Correlation with biological functions.

The frog skin-derived antimicrobial peptide esculentin-1a(1-21)NH2 [Esc(1-21)], and its diastereomer Esc(1-21)-1c (containing two D-amino acids at positions 14 and 17), have been recently found to neutralize the toxic effect of Pseudomonas aeruginosa lipopolysaccharide (LPS), although to different extents. Here, we studied the three-dimensional structure of both peptides in complex with P. aeruginosa LPS, by transferred nuclear Overhauser effect spectroscopy. Lack of NOE peaks revealed that both the peptides adopted a random coil structure in aqueous solution. However, Esc(1-21) adopted an amphipathic helical conformation in LPS micelles with 5 basic Lys residues forming a hydrophilic cluster. In comparison, the diastereomer maintained an alpha helical conformation only at the N-terminal region, whereas the C-terminal portion was quite flexible. Isothermal titration calorimetry (ITC) revealed that the interaction of Esc(1-21) with LPS is an exothermic process associated with a dissociation constant of -4µM. In contrast, Esc(1-21)-1c had almost 8 times weaker binding affinity to LPS micelles. Moreover, STD NMR data supported by docking analysis have identified those amino acid residues responsible for the peptide's binding to LPS micelles. Overall, the data provide important mechanistic insights on the interaction of esculentin-derived peptides with LPS and the reason for their different anti-endotoxin activity. These data might also assist to further design more potent antimicrobial peptides with antisepsis properties, which are highly needed to overcome the widespread concern of the available anti-infective agents.

Mapping out the intricate relationship of the HIV envelope protein and the membrane environment.

The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

The HIV gp41 pocket binding domain enables C-terminal heptad repeat transition from mediating membrane fusion to immune modulation.

For successful infection and propagation viruses must overcome many obstacles such as the immune system and entry into their host cells. HIV utilizes its trimeric envelope protein gp160, specifically the gp41 subunit, to enter its host cell. During this process, a gp41-central coiled coil is formed from three N- and three C-terminal heptad repeats, termed the six-helix bundle (SHB), which drives membrane fusion. Recently, T-cell suppression has been reported as an additional function for several regions of gp41 by interfering with the T-cell receptor (TCR) signalling cascade. One of these regions encompasses the conserved pocket binding domain (PBD) that is situated in the C-terminal heptad repeat (CHR) and stabilizes SHB formation. This could indicate that the PBD plays a role in T-cell suppression in addition to its role in membrane fusion. To investigate this dual function, we used two independent cell cultures coupled with biophysical techniques. The data reveal that the PBD mediates T-cell suppression by stabilizing a TCR-binding conformation in the membrane. Moreover, we show that the clinically used HIV fusion inhibitor T-20 did not show suppressive abilities, in contrast with the potent fusion inhibitor C34. In addition, by focusing on SHB conformation after its assembly, we shed light on a mechanism by which gp41's function alternates from membrane fusion facilitation to suppression of TCR activation.

The Transmembrane Domain of HIV-1 gp41 Inhibits T-Cell Activation by Targeting Multiple T-Cell Receptor Complex Components through Its GxxxG Motif.

To successfully infect and persist within its host, HIV-1 utilizes several immunosuppressive motifs within its gp41 envelope glycoprotein to manipulate and evade the immune system. The transmembrane domain (TMD) of gp41 downregulates T-cell receptor (TCR) signaling through a hitherto unknown mechanism. Interactions between TMDs within the membrane milieu have been shown to be typically mediated by particular amino acids, such as interactions between basic and acidic residues and dimerization motifs as GxxxG. The HIV-1 TMD exhibits both a polar arginine (Arg(696)) residue and a GxxxG motif, making them ideal candidates for mediators of TMD-TCR interaction. Using a primary T-cell activation assay and biochemical and biophysical methods, we demonstrate that the gp41 TMD directly interacts with TMDs of the TCR and the CD3 coreceptors (δ, γ, and ε) within the membrane, presumably leading to impairment of complex assembly. Additionally, we reveal that although Arg(696) does not affect TMD immunosuppression, the GxxxG motif is crucial in mediating gp41's TMD interaction with the CD3 coreceptors of the TCR. These findings suggest that compared with other gp41 immunosuppressive motifs, the gp41 TMD has multiple targets within the TCR complex, suggesting less susceptibility to evolutionary pressure and consequently being advantageous for the virus over the host immune response. Furthermore, as the GxxxG motif mediates interactions of the gp41 TMD with multiple receptors, it emerges as an attractive drug target. This multitarget inhibitory mechanism might be a strategy utilized by HIV to interfere with the function of additional host receptors.

Interfering with the Dimerization of the ErbB Receptors by Transmembrane Domain-Derived Peptides Inhibits Tumorigenic Growth in Vitro and in Vivo.

The ErbB family of tyrosine kinase receptors is a key element in preserving cell growth homeostasis. This family is comprised of four single-transmembrane domain proteins designated ErbB-1-4. Ligand binding initiates dimerization followed by tyrosine phosphorylation and signaling, which when uncontrolled lead to cancer. Accordingly, extensive research has been devoted to finding ErbB-intercepting agents, directed against ErbB-1 and ErbB-2, but so far, no inhibitor has targeted the transmembrane domain (TMD), which is instrumental for receptor dimerization and activation. Moreover, no antitumor agents targeted ErbB-3, which although it cannot generate signals in isolation, its heterodimerization with ErbB-2 leads to the most powerful and oncogenic signaling unit in the ErbB family. Here, to further elucidate the role of the interactions between the TMDs of the ErbB family in cancer, we investigated peptides derived from the TMDs of ErbB-1 and ErbB-2. We then focused on the C-terminal domains (B2C) and their analogue, named B2C-D, that contains both d- and l-amino acids. Both peptides incorporated the distal GXXXG dimerization motif to target the TMD of ErbB-3. Our results revealed that B2C-D is highly active both in vitro and in vivo. It significantly inhibits neuregulin- and EGF-induced ErbB activation, impedes the proliferation of a battery of human cancer cell lines, and retards tumor growth in vivo. Notably, combining low concentrations of B2C-D and gemcitabine chemotherapy completely arrested proliferation of pancreatic cancer cells. Biochemical and in vitro interaction studies suggest direct interference with the assembly of the wild-type ErbB-2-ErbB-3 heterodimer as the underlying mode of action. To the best of our knowledge, this is the first agent to target the TMDs of ErbB to delay tumor growth and signaling.

Defensive remodeling: How bacterial surface properties and biofilm formation promote resistance to antimicrobial peptides.

Multidrug resistance bacteria are a major concern worldwide. These pathogens cannot be treated with conventional antibiotics and thus alternative therapeutic agents are needed. Antimicrobial peptides (AMPs) are considered to be good candidates for this purpose. Most AMPs are short and positively charged amphipathic peptides, which are found in all known forms of life. AMPs are known to kill bacteria by binding to the negatively charged bacterial surface, and in most cases cause membrane disruption. Resistance toward AMPs can be developed, by modification of bacterial surface molecules, secretion of protective material and up-regulation or elimination of specific proteins. Because of the general mechanisms of attachment and action of AMPs, bacterial resistance to AMPs often involves biophysical and biochemical changes such as surface rigidity, cell wall thickness, surface charge, as well as membrane and cell wall modification. Here we focus on the biophysical, surface and surrounding changes that bacteria undergo in acquiring resistance to AMPs. In addition we discuss the question of whether bacterial resistance to administered AMPs might compromise our innate immunity to endogenous AMPs. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.

Esculentin-1a-Derived Peptides Promote Clearance of Pseudomonas aeruginosa Internalized in Bronchial Cells of Cystic Fibrosis Patients and Lung Cell Migration: Biochemical Properties and a Plausible Mode of Action.

Pseudomonas aeruginosa is the major microorganism colonizing the respiratory epithelium in cystic fibrosis (CF) sufferers. The widespread use of available antibiotics has drastically reduced their efficacy, and antimicrobial peptides (AMPs) are a promising alternative. Among them, the frog skin-derived AMPs, i.e., Esc(1-21) and its diastereomer, Esc(1-21)-1c, have recently shown potent activity against free-living and sessile forms of P. aeruginosa Importantly, this pathogen also escapes antibiotics treatment by invading airway epithelial cells. Here, we demonstrate that both AMPs kill Pseudomonas once internalized into bronchial cells which express either the functional or the ΔF508 mutant of the CF transmembrane conductance regulator. A higher efficacy is displayed by Esc(1-21)-1c (90% killing at 15 µM in 1 h). We also show the peptides' ability to stimulate migration of these cells and restore the induction of cell migration that is inhibited by Pseudomonas lipopolysaccharide when used at concentrations mimicking lung infection. This property of AMPs was not investigated before. Our findings suggest new therapeutics that not only eliminate bacteria but also can promote reepithelialization of the injured infected tissue. Confocal microscopy indicated that both peptides are intracellularly localized with a different distribution. Biochemical analyses highlighted that Esc(1-21)-1c is significantly more resistant than the all-l peptide to bacterial and human elastase, which is abundant in CF lungs. Besides proposing a plausible mechanism underlying the properties of the two AMPs, we discuss the data with regard to differences between them and suggest Esc(1-21)-1c as a candidate for the development of a new multifunctional drug against Pseudomonas respiratory infections.

The HIV-1 envelope transmembrane domain binds TLR2 through a distinct dimerization motif and inhibits TLR2-mediated responses.

HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

Mechanisms of biofilm inhibition and degradation by antimicrobial peptides.

Many bacteria live as biofilms to cope with unfavourable surroundings. Biofilms start from (i) a planktonic stage, (ii) initial adhesion to surfaces and (iii) formation of sessile micro-colonies that secrete extracellular polymeric substance (EPS), leading to bacterial resistance to antibiotics. Antimicrobial peptides (AMPs) are extensively studied with regard to planktonic bacteria but much less so with regard to biofilm formation. In the present study, we investigated how the above three steps are affected by the properties of the AMPs using a series of peptides composed of six lysines and nine leucines, which differ in their sequences and hence their biophysical properties. Treatment with bactericidal peptides at non-inhibitory concentrations resulted in reduced biofilm growth, for some starting from 25 nM which is 0.2 and 0.4% of their minimum inhibitory concentration (MIC 6.3 and 12.5 µM, respectively), continuing in a dose-dependent manner. We suggest that reduced bacterial adhesion to surfaces and decreased biofilm growth are due to the peptide's ability to coat either the biomaterial surface or the bacterium itself. Degradation of established biofilms by bactericidal and non-bactericidal peptides, within 1 h of incubation, occurs by either killing of embedded bacteria or detachment of live ones. In addition to shedding light on the mechanism of biofilm inhibition and degradation, these data may assist in the design of anti-biofilm AMPs.