Thermodynamics of RTA3 peptide binding to membranes and consequences for antimicrobial activity

RTA3 is an α-helical, amphipathic peptide with broad-spectrum activity against Gram-negative bacteria and low mammalian cell toxicity. RTA3 contains a cysteine residue, replacement of which with an alanine or serine (RTA3-C15S) virtually abolishes antimicrobial activity. Much of the activity of RTA3 can be recovered in RTA3-C15L, indicating that the C15 residue functions largely as a bulky hydrophobic side chain promoting target cell membrane interactions. The poorly active RTA3-C15S is a useful variant for assessing the mechanistic aspects of RTA3 activity. Binding and membrane perturbation in vesicles containing different proportions of negative surface charge are analyzed in terms of amino acid-specific free energy contributions to interfacial binding, which likely underlie variations in antimicrobial activity amongst RTA3 variants. Comparison with published free energy scales indicates that the reduced electrostatic contribution to binding to membranes having reduced negative surface charge can be compensated in RTA3 (but not RTA3-C15S) by a slightly deeper insertion of the C-terminus of the peptide to maximize hydrophobic contributions to binding. Analysis of inner membrane (IM)- and outer membrane (OM)-selective permeabilization of Escherichiacoli demonstrates a broad similarity between peptide effects on vesicles with low negative surface charge (20% negatively charged lipids), E.coli membrane perturbation, and antimicrobial activity, supporting a role for membrane perturbation in the killing mechanism of RTA3. The results demonstrate that large variations in antimicrobial activity on subtle changes in amino acid sequence in helical amphipathic peptides can be rationalized in terms of the thermodynamics of peptide binding to membranes, allowing a more systematic understanding of antimicrobial activity in these peptides.     

Can antimicrobial peptides scavenge around a cell in less than a second?

Antimicrobial peptides, which play multiple host-defense roles, have garnered increased experimental focus because of their potential applications in the pharmaceutical and food production industries. While their mechanisms of action are richly debated, models that have been advanced share modes of peptide-lipid interactions that require peptide dynamics. Before the highly cooperative and specific events suggested in these models take place, peptides must undergo an important process of migration along the membrane surface and delivery from their site of binding on the membrane to the actual site of functional performance. This phenomenon, which contributes significantly to antimicrobial function, is poorly understood, largely due to a lack of experimental and computational tools needed to assess it. Here, we use ¹⁵N solid-state nuclear magnetic resonance to obtain molecular level data on the motions of piscidin's amphipathic helices on the surface of phospholipid bilayers. The studies presented here may help contribute to a better understanding of the speed at which the events that lead to antimicrobial response take place. Specifically, from the perspective of the kinetics of cellular processes, we discuss the possibility that piscidins and perhaps many other amphipathic antimicrobial peptides active on the membrane surface may represent a class of fast scavengers rather than static polypeptides attached to the water-lipid interface.     

Resistance to ciprofloxacin by enhancement of antioxidant defenses in biofilm and planktonic Proteus mirabilis

Antibiotic resistance and antioxidant defense were induced by ciprofloxacin in planktonic Proteus mirabilis and compared with the natural antibiotic resistance of biofilm. Resistant variants (1X and 1Y) were obtained from cultures of the sensitive wild type “wt” strain 1 in the presence of the antibiotic. Planktonic strain 1 exhibited oxidative stress with increases in the reactive oxygen species (ROS) and consumption of NO in the presence of ciprofloxacin, whereas 1X and 1Y suffered non-significant rises in ROS generation, but produced and consumed more NO than sensitive strain 1. The two resistant variants were more resistant to telluride than wt and showed increased levels of intracellular superoxide dismutase (SOD) and glutathione (GSH). However, ciprofloxacin did not stimulate oxidative stress in biofilm. The production of ROS and NO with or without ciprofloxacin was less significant in biofilms than in an equivalent number of planktonic bacteria; sensitive and resistant strains did not present differences. On the other hand, SOD and GSH were more elevated in the biofilm than in planktonic bacteria. In summary, these results indicate that ciprofloxacin can induce resistance by the enhancement of antioxidant defense in planktonic bacteria, similar to the natural resistance occurring in biofilm. This feature may be added to the factors that regulate the susceptibility to this antibiotic.     

Level of M(IP)2C sphingolipid affects plant defensin sensitivity, oxidative stress resistance and chronological life-span in yeast

The antifungal plant defensin DmAMP1 interacts with fungal sphingolipids of mannosyldiinositolphosphorylceramide (M(IP)2C) class. We screened a Saccharomyces cerevisiae transposon (Tn) mutant library against DmAMP1 and identified one DmAMP1-resistant mutant with the Tn inserted in the M(IP)2C biosynthesis gene IPT1 (DmTn11) and one DmAMP1-hypersensitive mutant with the Tn inserted in rDNA (HsTnII). However, tetrad analysis pointed to HsTnII as a spontaneous mutant. Apparently, membranes of DmTn11 lack M(IP)2C, whereas membranes of HsTnII have increased M(IP)2C levels. In addition, DmTn11 and HsTnII are characterized by increased and reduced oxidative stress resistance/chronological life-span (CL), respectively. A putative involvement of M(IP)2C in oxidative stress and CL in yeast is discussed.     

Antifungal effect and pore-forming action of lactoferricin B like peptide derived from centipede Scolopendra subspinipes mutilans

The centipede Scolopendra subspinipes mutilans has been a medically important arthropod species by using it as a traditional medicine for the treatment of various diseases. In this study, we derived a novel lactoferricin B like peptide (LBLP) from the whole bodies of adult centipedes, S. s. mutilans, and investigated the antifungal effect of LBLP. LBLP exerted an antifungal and fungicidal activity without hemolysis. To investigate the antifungal mechanism of LBLP, a membrane study with propidium iodide was first conducted against Candida albicans. The result showed that LBLP caused fungal membrane permeabilization. The assays of the three dimensional flow cytometric contour plot and membrane potential further showed cell shrinkage and membrane depolarization by the membrane damage. Finally, we confirmed the membrane-active mechanism of LBLP by synthesizing model membranes, calcein and FITC-dextran loaded large unilamellar vesicles. These results showed that the antifungal effect of LBLP on membrane was due to the formation of pores with radii between 0.74 nm and 1.4 nm. In conclusion, this study suggests that LBLP exerts a potent antifungal activity by pore formation in the membrane, eventually leading to fungal cell death.     

Determining the effect of the incorporation of unnatural amino acids into antimicrobial peptides on the interactions with zwitterionic and anionic membrane model systems

Circular Dichroism (CD), isothermal calorimetry (ITC) and calcein fluorescence leakage experiments were conducted to provide insight into the mechanisms of binding of a series of antimicrobial peptides containing unnatural amino acids (Ac-XF-Tic-Oic-XK-Tic-Oic-XF-Tic-Oic-XK-Tic-KKKK-CONH₂) to zwitterionic and anionic micelles, SUVs and LUVs; where X (Spacer# 1) is either Gly, β-Ala, Gaba or 6-aminohexanoic acid. It is the intent of this investigation to correlate these interactions with the observed potency and selectivity against several different strains of bacteria. The CD spectra of these compounds in the presence of zwitterionic DPC micelles and anionic SDS micelles are very different indicating that these compounds adopt different conformations on binding to the surface of anionic and zwitterionic membrane models. These compounds also exhibited very different CD spectra in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG SUVs and LUVs, indicating the formation of different conformations on interaction with the two membrane types. This observation is also supported by ITC and calcein leakage data. ITC data suggested these peptides interact primarily with the surface of zwitterionic LUVs and was further supported by fluorescence experiments where the interactions do not appear to be concentration dependent. In the presence of anionic membranes, the interactions appear more complex and the calorimetric and fluorescence data both imply pore formation is dependent on peptide concentration. Furthermore, evidence suggests that as the length of Spacer# 1 increases the mechanism of pore formation also changes. Based on the observed differences in the mechanisms of interactions with zwitterionic and anionic LUVs these AMPs are potential candidates for further drug development.     

Error-prone and error-restrictive mutations affecting ribosomal protein S12

Ribosomal protein S12 plays a pivotal role in decoding functions on the ribosome. X-ray crystallographic analyses of ribosomal complexes have revealed that S12 is involved in the inspection of codon-anticodon pairings in the ribosomal A site, as well as in the succeeding domain rearrangements of the 30S subunit that are essential for accommodation of aminoacyl-tRNA. A role for S12 in tRNA selection is also well supported by classical genetic analyses; mutations affecting S12 are readily isolated in bacteria and organelles, since specific alterations in S12 confer resistance to the error-inducing antibiotic streptomycin, and the ribosomes from many such streptomycin-resistant S12 mutants display decreased levels of miscoding. However, substitutions that confer resistance to streptomycin likely represent a very distinct class of all possible S12 mutants. Until recently, the technical difficulties in generating random, unselectable mutations in essential genes in complex operons have generally precluded the analysis of other classes of S12 alterations. Using a recombineering approach, we have targeted the Escherichia coli rpsL gene, encoding S12, for random mutagenesis and screened the resulting mutants for effects on decoding fidelity. We have recovered over 40 different substitutions located throughout the S12 protein that alter the accuracy of translation without substantially affecting the sensitivity to streptomycin. Moreover, this collection includes mutants that promote miscoding, as well as those that restrict decoding errors. These results affirm the importance of S12 in decoding processes and indicate that alterations in this essential protein can have diverse effects on the accuracy of decoding.     

Folding of AcrB Subunit Precedes Trimerization

AcrB and its homologues are major players in the efflux of anti-microbials out of Gram-negative bacteria. The structural and functional unit of AcrB is a homo-trimer. The assembly process of obligate membrane protein oligomers, including AcrB, remains elusive. It is not clear if an individual subunit folds into a monomeric form first followed by association (three-stage pathway) or if association occurs simultaneously with subunit folding (two-stage pathway). To answer this question, we investigated the feasibility of creating a folded monomeric AcrB mutant. The existence of well-folded monomers in the cell membrane would be an evidence of a three-stage pathway. A monomeric AcrB mutant, AcrB_Δloop, was created through the truncation of a protruding loop that appeared to contribute to the stability of an AcrB trimer. AcrB_Δloop expressed at a level similar to that of wild-type AcrB. The secondary structure content and tertiary conformation of AcrB_Δloop were very similar to those of wild-type AcrB. However, when expressed in an acrB-deficient strain, AcrB_Δloop failed to complement its defect in drug efflux. Results from blue native polyacrylamide gel electrophoresis and chemical cross-linking experiments suggested that AcrB_Δloop existed as a monomer. The expression of this monomeric mutant in a wild-type Escherichia coli strain did not have a significant dominant-negative effect, suggesting that the mutant could not effectively co-assemble with genomic AcrB. AcrB_Δloop is the first monomeric mutant reported for the intrinsically trimeric AcrB. The structural characterization results of this mutant suggest that the oligomerization of AcrB occurs through a three-stage pathway involving folded monomers.     

Comparison of disc diffusion, Etest and agar dilution for susceptibility testing of colistin against Enterobacteriaceae

Aims: In this study, we compared different methods of colistin susceptibility testing, disc diffusion, agar dilution and Etest using a set of Enterobacteriaceae isolates that included colistin‐resistant strains. Methods and results: Susceptibility of 200 clinical isolates of Enterobacteriaceae to colistin was tested to compare agar dilution (reference method), disc diffusion (50 and 10 μg) and Etest. MICs (minimum inhibitory concentrations) were interpreted using the criteria established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Colistin exhibited excellent activity against Escherichia coli and E. cloacae (MIC₉₀ = 0·5 mg l^?1). In contrast, colistin was less active against Klebsiella pneumoniae (MIC₉₀ = 16 mg l^?1). Resistance rates varied from 0% in E. coli to 1·8% in E. cloacae and 13% in K. pneumoniae. High rates of very major errors were observed in the disc diffusion test using either the criteria of the Comité de l’antibiogramme de la Société Française de Microbiologie (CA‐SFM) or the criteria of the Clinical and Laboratory Standards Institute (CLSI), respectively, 3·5 and 2·5%. When the criteria of Gales et al. were applied, the number of very major errors was reduced to one (0·5%). The Etest showed good concordance with agar dilution method. Conclusion: Disc susceptibility testing methods are unreliable on detecting colistin resistance. MIC should be determined to confirm the susceptibility results by disc diffusion. Significance and Impact of the study: We recommend the determination of MIC by Etest for all multidrug‐resistant Enterobacteriaceae when colistin is required for the treatment.     

RYH: a minimal peptidic sequence obtained from beta-chain hemoglobin exhibiting an antimicrobial activity

Bovine hemoglobin is an animal protein described as source of bioactive peptides. Enzymatic hydrolysis of this protein results into some peptides exhibiting antimicrobial activity against Gram-positive and Gram-negative bacteria. In this study, a family of peptides from the beta chain (beta-114-145 derived peptides) obtained by peptic hydrolysis of bovine hemoglobin, was purified by reverse-phase HPLC and characterized by different analytical techniques (mass spectrometry, circular dichroism). The minimum inhibitory concentration was determined to show the antimicrobial activity of these peptides. Four bacterial strains were used: two Gram-negative (Escherichia coli and Salmonella Enteritidis) and two Gram-positive strains (Listeria innocua and Micrococcus luteus). The effect of these peptides on artificial membrane was also measured. Our findings showed that the peptide β114-145 and its peptic derivatives contain the RYH sequence. The most antimicrobial peptide is the RYH peptide which was the shortest one.