Helix induction in antimicrobial peptides by alginate in biofilms

Bacterial exopolysaccharides provide protection against phagocytosis, opsonization, and dehydration and act as a major structural component of the extracellular matrix in biofilms. They contribute to biofilm-related resistance by acting as a diffusion barrier to positively charged antimicrobial agents including cationic antimicrobial peptides (CAPs). We previously created novel CAPs consisting of a nonamphipathic hydrophobic core flanked by Lys residues and containing a Trp residue in the hydrophobic segment as a fluorescent probe. Peptides of this type above a specific hydrophobicity threshold insert spontaneously into membranes and have antimicrobial activity against Gram-positive and Gram-negative bacteria at micromolar concentrations. Here we show that alginate, a polymer of beta-d-mannuronate and alpha-l-guluronate secreted by the cystic fibrosis pathogen Pseudomonas aeruginosa, induces an alpha-helical conformation detected by circular dichroism spectroscopy and blue shifts in Trp fluorescence maxima in peptides above the hydrophobicity threshold, changes typically observed upon association of such peptides with nonpolar (membrane) environments. Parallel effects were observed in the archetypical CAPs magainin II amide and cecropin P1. Fluorescence resonance energy transfer studies indicated that alginate induces peptide-peptide association only in peptides above the hydrophobicity threshold, suggesting that the hydrophilic alginate polymer behaves as an "auxiliary membrane" for the bacteria, demonstrating a unique protective role for biofilm matrices against CAPs.     

Membrane interactions of designed cationic antimicrobial peptides: the two thresholds

Novel cationic antimicrobial peptides (CAPs) designed in our lab-typified by sequences such as KKKKKKAAX-AAXAAXAA-NH(2), where X = Phe/Trp-display high antibacterial activity but exhibit little or no hemolytic activity towards human red blood cells even at high doses. To clarify the mechanism of their selectivity for bacterial versus mammalian membranes and to increase our understanding of the relationships between primary sequence and bioactivity, a library of derivatives was prepared by increasing segmental hydrophobicity, in which systematic substitutions of Ala for two, three, or four Leu residues were made. Conformationally constrained dimeric and cyclic derivatives were also synthesized. The peptides were examined for activity against pathogenic bacteria (Pseudomonas aeruginosa), hemolytic activity on human red blood cells, and insertion into models of natural bacterial membranes (containing anionic lipids) and mammalian membranes (containing zwitterionic lipids + cholesterol). Results were compared with corresponding properties of the natural CAPs magainin and cecropin. Using circular dichroism and fluorescence spectroscopy, we found that peptide conformation and membrane insertion were sequence dependent, both upon the number of Leu residues, and upon their positions along the hydrophobic core. Membrane disruption was likely enhanced by the fact that the peptides contain potent dimerization-promoting sequence motifs, as assessed by SDS-PAGE gel analysis. The overall results led us to identify distinctions in the mechanism of actions of these CAPs for disruption of bacterial versus mammalian membranes, the latter dependent on surpassing a "second hydrophobicity threshold" for insertion into zwitterionic membranes.     

Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions

Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in many organisms, including humans, and offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death. In this work, we studied the biophysical and microbiological characteristics of designed CAPs varying in hydrophobicity levels and charge distributions by a variety of biophysical and biochemical approaches, including in-tandem atomic force microscopy, attenuated total reflection-FTIR, CD spectroscopy, and SDS-PAGE. Peptide structural properties were correlated with their membrane-disruptive abilities and antimicrobial activities. In bacterial lipid model membranes, a time-dependent increase in aggregated β-strand-type structure in CAPs with relatively high hydrophobicity (such as KKKKKKALFALWLAFLA-NH(2)) was essentially absent in CAPs with lower hydrophobicity (such as KKKKKKAAFAAWAAFAA-NH(2)). Redistribution of positive charges by placing three Lys residues at both termini while maintaining identical sequences minimized self-aggregation above the dimer level. Peptides containing four Leu residues were destructive to mammalian model membranes, whereas those with corresponding Ala residues were not. This finding was mirrored in hemolysis studies in human erythrocytes, where Ala-only peptides displayed virtually no hemolysis up to 320 μM, but the four-Leu peptides induced 40-80% hemolysis at the same concentration range. All peptides studied displayed strong antimicrobial activity against Pseudomonas aeruginosa (minimum inhibitory concentrations of 4-32 μM). The overall findings suggest optimum routes to balancing peptide hydrophobicity and charge distribution that allow efficient penetration and disruption of the bacterial membranes without damage to mammalian (host) membranes.     

Folding amphipathic helices into membranes: amphiphilicity trumps hydrophobicity

High amphiphilicity is a hallmark of interfacial helices in membrane proteins and membrane-active peptides, such as toxins and antimicrobial peptides. Although there is general agreement that amphiphilicity is important for membrane-interface binding, an unanswered question is its importance relative to simple hydrophobicity-driven partitioning. We have examined this fundamental question using measurements of the interfacial partitioning of a family of 17-residue amidated-acetylated peptides into both neutral and anionic lipid vesicles. Composed only of Ala, Leu, and Gln residues, the amino acid sequences of the peptides were varied to change peptide amphiphilicity without changing total hydrophobicity. We found that peptide helicity in water and interface increased linearly with hydrophobic moment, as did the favorable peptide partitioning free energy. This observation provides simple tools for designing amphipathic helical peptides. Finally, our results show that helical amphiphilicity is far more important for interfacial binding than simple hydrophobicity.     

Pleurocidin-derived antifungal peptides with selective membrane-disruption effect

Pleurocidin (Ple) is a peptide derived from the winter flounder. In our previous study, we reported the antifungal effect of Ple and its mode of action. To develop novel antifungal peptides useful as therapeutic agents, two analogs, with amino acid substitutions, were designed to decrease the net hydrophobicity by Arg (R) or Ser (S)-substitution at the hydrophobic face of Ple without changing the amphipathic structure. By substituting Ser, the hydrophobicity of the peptide (anal-S) was decreased, and by substituting Arg, though the hydrophobicity of the peptide (anal-R) was decreased, the cationicity of this peptide was increased. CD measurements showed the substitution of Arg or Ser decrease the α-helical conformation of analog peptides. Studies with analog peptides have shown decreases in hydrophobicity and α-helicity do not affect antifungal activity but decrease hemolytic activity. These results suggest that highly hydrophobic and α-helical natures are not desirable in the design of antimicrobial peptides.     

Temporin-SHf,a New Type of Phe-rich and Hydrophobic Ultrashort Antimicrobial Peptide

Because issues of cost and bioavailability have hampered the development of gene-encoded antimicrobial peptides to combat infectious diseases, short linear peptides with high microbial cell selectivity have been recently considered as antibiotic substitutes. A new type of short antimicrobial peptide, designated temporin-SHf, was isolated and cloned from the skin of the frog Pelophylax saharica. Temporin-SHf has a highly hydrophobic sequence (FFFLSRIFa) and possesses the highest percentage of Phe residues of any known peptide or protein. Moreover, it is the smallest natural linear antimicrobial peptide found to date, with only eight residues. Despite its small size and hydrophobicity, temporin-SHf has broad-spectrum microbicidal activity against Gram-positive and Gram-negative bacteria and yeasts, with no hemolytic activity. CD and NMR spectroscopy combined with restrained molecular dynamics calculations showed that the peptide adopts a well defined non-amphipathic α-helical structure from residue 3 to 8, when bound to zwitterionic dodecyl phosphocholine or anionic SDS micelles. Relaxation enhancement caused by paramagnetic probes showed that the peptide adopts nearly parallel orientations to the micelle surface and that the helical structure is stabilized by a compact hydrophobic core on one face that penetrates into the micelle interior. Differential scanning calorimetry on multilamellar vesicles combined with membrane permeabilization assays on bacterial cells indicated that temporin-SHf disrupts the acyl chain packing of anionic lipid bilayers, thereby triggering local cracks and microbial membrane disintegration through a detergent-like effect probably via the carpet mechanism. The short length, compositional simplicity, and broad-spectrum activity of temporin-SHf make it an attractive candidate to develop new antibiotic agents.     

Structural interrogation of phosphoproteome identified by mass spectrometry reveals allowed and disallowed regions of phosphoconformation

Background

Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics.

Results

The binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs.

Conclusions

The biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.     

Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function

During the course of chronic cystic fibrosis (CF) infections, Pseudomonas aeruginosa undergoes a conversion to a mucoid phenotype, which is characterized by overproduction of the exopolysaccharide alginate. Chronic P. aeruginosa infections involve surface-attached, highly antibiotic-resistant communities of microorganisms organized in biofilms. Although biofilm formation and the conversion to mucoidy are both important aspects of CF pathogenesis, the relationship between them is at the present unclear. In this study, we report that the overproduction of alginate affects biofilm development on an abiotic surface. Biofilms formed by an alginate-overproducing strain exhibit a highly structured architecture and are significantly more resistant to the antibiotic tobramycin than a biofilm formed by an isogenic nonmucoid strain. These results suggest that an important consequence of the conversion to mucoidy is an altered biofilm architecture that shows increasing resistance to antimicrobial treatments.     

Correlation between the activities of alpha-helical antimicrobial peptides and hydrophobicities represented as RP HPLC retention times

PTP7 is a 13-amino acid residue peptide designed from gaegurin 6, an antimicrobial peptide isolated from skin secretions of Rana rugosa. In order to examine the effect of hydrophobicity on antimicrobial activity, a series of PTP7 derivatives were constructed and analyzed the activity against bacteria and artificial membrane. We found that the mean hydrophobicity by simple summation of hydrophobicity of each constituent amino acid did not necessarily describe the hydrophobic property of antimicrobial peptides. The mean hydrophobicity did not show close correlation with the observed hydrophobicity by measuring reverse phase high performance liquid chromatography (RP HPLC) retention time. The observed hydrophobicity represented as RP HPLC retention time correlated well with the activity against artificial membrane and Gram positive bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, rather than mean hydrophobicity. However, antimicrobial activity against Gram negative bacteria, such as Escherichia coli, did not show correlation with RP HPLC retention time. These data indicate that the RP HPLC retention time should be exploited rather than the mean hydrophobicity in the analysis of the relationship between hydrophobicity and antimicrobial activity.     

Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 degrees C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.     

A molecular mechanism for lipopolysaccharide protection of Gram-negative bacteria from antimicrobial peptides

Cationic antimicrobial peptides serve as the first chemical barrier between all organisms and microbes. One of their main targets is the cytoplasmic membrane of the microorganisms. However, it is not yet clear why some peptides are active against one particular bacterial strain but not against others. Recent studies have suggested that the lipopolysaccharide (LPS) outer membrane is the first protective layer that actually controls peptide binding and insertion into Gram-negative bacteria. In order to shed light on these interactions, we synthesized and investigated a 12-mer amphipathic alpha-helical antimicrobial peptide (K(5)L(7)) and its diastereomer (4D-K(5)L(7)) (containing four d-amino acids). Interestingly, although both peptides strongly bind LPS bilayers and depolarize bacterial cytoplasmic membranes, only the diastereomer kills Gram-negative bacteria. Attenuated total reflectance Fourier transform infrared, CD, and surface plasmon resonance spectroscopies revealed that only the diastereomer penetrates the LPS layer. In contrast, K(5)L(7) binds cooperatively to the polysaccharide chain and the outer phosphate groups. As a result, the self-associated K(5)L(7) is unable to traverse through the tightly packed LPS molecules, revealed by epifluorescence studies with LPS giant unilamellar vesicles. The difference in the peptides' modes of binding is further demonstrated by the ability of the diastereomer to induce LPS miscellization, as shown by transmission electron microscopy. In addition to increasing our understanding of the molecular basis of the protection of bacteria by LPS, this study presents a potential strategy to overcome resistance by LPS, and it should help in the design of antimicrobial peptides for future therapeutic purposes.     

The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing

The ability of Pseudomonas aeruginosa to form biofilms and cause chronic infections in the lungs of cystic fibrosis patients is well documented. Numerous studies have revealed that P. aeruginosa biofilms are highly refractory to antibiotics. However, dramatically fewer studies have addressed P. aeruginosa biofilm resistance to the host's immune system. In planktonic, unattached (nonbiofilm) P. aeruginosa, the exopolysaccharide alginate provides protection against a variety of host factors yet the role of alginate in protection of biofilm bacteria is unclear. To address this issue, we tested wild-type strains PAO1, PA14, the mucoid cystic fibrosis isolate, FRD1 (mucA22+), and the respective isogenic mutants which lacked the ability to produce alginate, for their susceptibility to human leukocytes in the presence and absence of IFN-gamma. Human leukocytes, in the presence of recombinant human IFN-gamma, killed biofilm bacteria lacking alginate after a 4-h challenge at 37 degrees C. Bacterial killing was dependent on the presence of IFN-gamma. Killing of the alginate-negative biofilm bacteria was mediated through mononuclear cell phagocytosis since treatment with cytochalasin B, which prevents actin polymerization, inhibited leukocyte-specific bacterial killing. By direct microscopic observation, phagocytosis of alginate-negative biofilm bacteria was significantly increased in the presence of IFN-gamma vs all other treatments. Addition of exogenous, purified alginate to the alginate-negative biofilms restored resistance to human leukocyte killing. Our results suggest that although alginate may not play a significant role in bacterial attachment, biofilm development, and formation, it may play an important role in protecting mucoid P. aeruginosa biofilm bacteria from the human immune system.     

Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation

Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents. Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute. Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P. aeruginosa strains PAO1 and PA14. We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development. Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome. Reverse genetics was employed to generate mutations in genes from these clusters. We discovered that one group of genes, designated psl, are important for biofilm initiation. A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays. This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis. These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix. Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P. aeruginosa in cystic fibrosis and other infections involving biofilms.     

Role of helicity of α-helical antimicrobial peptides to improve specificity

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Grampositive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.     

抗菌肽的抗生物膜机理研究进展

生物膜,也称为生物被膜,是指附着于有生命或无生命物体表面被细菌胞外大分子包裹的有组织的细菌群体。与浮游菌相比,生物膜内的细菌对抗生素的耐受性提高了10–1000倍,是造成目前细菌耐药的主要原因之一。作为一种新型抗菌制剂,抗菌肽的使用为生物膜感染的治疗提供了一种新的思路和手段。抗菌肽在抑制生物膜形成、杀灭生物膜内细菌以及消除成熟生物膜的过程中发挥了独特的优势。文中分析了近30年的数据,从细菌生物膜的结构入手,对抗菌肽可能的抗生物膜机理进行了综述,以期为抗菌肽临床治疗生物膜感染提供一定参考。     

Mode of action of membrane active antimicrobial peptides

Water-membrane soluble protein and peptide toxins are used in the defense and offense systems of all organisms, including plants and humans. A major group includes antimicrobial peptides, which serve as a nonspecific defense system that complements the highly specific cell-mediated immune response. The increasing resistance of bacteria to conventional antibiotics stimulated the isolation and characterization of many antimicrobial peptides for potential use as new target antibiotics. The finding of thousands of antimicrobial peptides with variable lengths and sequences, all of which are active at similar concentrations, suggests a general mechanism for killing bacteria rather than a specific mechanism that requires preferred active structures. Such a mechanism is in agreement with the "carpet model" that does not require any specific structure or sequence. It seems that when there is an appropriate balance between hydrophobicity and a net positive charge the peptides are active on bacteria. However, selective activity depends also on other parameters, such as the volume of the molecule, its structure, and its oligomeric state in solution and membranes. Further, although many studies support that bacterial membrane damage is a lethal event for bacteria, other studies point to a multihit mechanism in which the peptide binds to several targets in the cytoplasmic region of the bacteria.     

Effects of Hydrophobic Amino Acid Substitutions on Antimicrobial Peptide Behavior

Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.     

Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua

A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism.     

The protective layer of biofilm: a repellent function for a new class of amphiphilic proteins

Bacteria can survive harsh conditions when growing in complex communities of cells known as biofilms. The matrix of the biofilm presents a scaffold where cells are attached to each other and to the surface. The biofilm matrix is also a protective barrier that confers tolerance against various antimicrobial agents. In this issue of Molecular Microbiology, Kobayashi and Iwano (2012) show that the liquid permeability of Bacillus subtilis biofilms is determined by a small secreted protein, i.e. BslA (formerly called YuaB). BslA is important for the proper development of biofilms, but unlike exopolysaccharide and TasA, is not directly involved in cell cluster formation, and is synthesized following the production of exopolysaccharide and amyloid fibres. The amphiphilic BslA protein forms a polymer in vitro and localizes in vivo to the surface of the biofilm. The microstructures of the biofilm wrinkles are reduced in the bslA mutant strain and the liquid repellency of the biofilm surface is diminished. Exogenously added BslA(42-181) protein complements the bslA mutation and restores not only water repellency, but also the formation of aerial structures. This study demonstrates that amphiphilic proteins have an important role in liquid repellency of biofilms and it suggests that these polymers contribute to antimicrobial resistance.     

Modified Cysteine-Deleted Tachyplesin (CDT) Analogs as Linear Antimicrobial Peptides: Influence of Chain Length,Positive Charge,and Hydrophobicity on Antimicrobial and Hemolytic Activity

Due to increasing resistance of bacteria to traditional antibiotics, antimicrobial peptides are being investigated as a promising alternative. Tachyplesin, an antimicrobial peptide isolated from horseshoe crab, inhibits the growth of many different types of bacteria with its ability to permeabilize the cell membrane. Starting with a previously reported linear tachyplesin analog lacking cysteine (cysteine-deleted tachyplesin, CDT, KWFRVYRGIYRRR-CONH₂), this study examines the systematic deletion of the C-terminal arginines and the N-terminal lysine, addition of positively charged N-and C-terminal residues, replacement of arginine with similarly-charged lysine, and replacement of hydrophobic residues with aliphatic, aromatic, fluoro-substituted aromatic, and bicyclic amino acids to examine effects on activity. The 16 modified CDT analogs were tested for their ability to disrupt model liposomes, and minimum inhibitory concentrations were determined for gram-positive and gram-negative bacterial strains. Hemolytic activity also was assessed. Overall, results indicate that elimination of two C-terminal arginine residues results in a peptide ([des-Arg^12,13]CDT) with preserved antimicrobial activity but a reduction in hemolysis, a selectivity desirable for a therapeutic agent. Additional deletion was not tolerated, nor was addition of residues at the termini. Analysis of the 16 analogs also reveals the importance of hydrophobicity, not necessarily aromaticity, as an analog with hydrophobic isoleucine residues placed throughout the sequence ([Ile^2,3,6,10]CDT) displayed comparable antimicrobial activity to CDT with lower hemolysis, representing a promising antimicrobial peptide with lowered toxicity.