Lipid-Induced Organization of a Primary Amphipathic Peptide: A Coupled AFM-Monolayer Study

To better understand the nature of the mechanism involved in the membrane uptake of a vector peptide, the interactions between dioleoylphosphatidylcholine and a primary amphipathic peptide containing a signal peptide associated with a nuclear localization sequence have been studied by isotherms analysis of mixed monolayers spread at the air-water interface. The peptide and the lipid interact through strong hydrophobic interactions with expansion of the mean molecular area that resulted from a lipid-induced modification of the organization of the peptide at the interface. In addition, a phase separation occurs for peptide molar fraction ranging from about 0.08 to 0.4 Atomic force microscopy observations made on transferred monolayers confirm the existence of phase separation and further reveal that mixed lipid-peptide particles are formed, the size and shape of which depend on the peptide molar fraction. At low peptide contents, round-shaped particles are observed and an increase of the peptide amount, simultaneously to the lipidic phase separation, induces morphological changes from bowls to filamentous particles. Fourier transform infrared spectra (FTIR) obtained on transferred monolayers indicate that the peptide adopts a β-like structure for high peptide molar fractions. Such an approach involving complementary methods allows us to conclude that the lipid and the peptide have a nonideal miscibility and form mixed particles which phase separate. Received: 31 July 1998/Revised: 4 November 1998     

Isomerization of an Antimicrobial Peptide Broadens Antimicrobial Spectrum to Gram-Positive Bacterial Pathogens

The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.     

Outer Membrane Protein I of Pseudomonas aeruginosa Is a Target of Cationic Antimicrobial Peptide/Protein

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and α-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and α-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric α-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of α-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

中国林蛙皮肤抗菌肽抗菌的特性

从林蛙皮肤中分离到具有抗菌活性的多肽混合物——多肽FⅢ。抑菌实验表明,林蛙皮肤中小分子活性肽对革兰氏阳性细菌、革兰氏阴性细菌都具有一定的抗菌作用,并且此粗提物的抗菌活性远远高于传统食品防腐剂苯甲酸钠和山梨酸钾的抗菌活性。     

Insights into Mechanisms and Proteomic Characterisation of Pseudomonas aeruginosa Adaptation to a Novel Antimicrobial Substance

Antibiotic resistance has been reported since the introduction of synthetic antibiotics. Bacteria, such as one of the most common nosocomial pathogens P. aeruginosa, adapt quickly to changing environmental conditions, due to their short generation time. Thus microevolutional changes can be monitored in situ. In this study, the microevolutional process of Pseudomonas aeruginosa PAO1 resistance against a recently developed novel antibacterial zinc Schiff-base (ZSB) was investigated at the proteome level. After extended exposure to ZSB the passaged strain differed in tolerance against ZSB, with the adapted P. aeruginosa PAO1 exhibiting 1.6 times higher minimal inhibitory concentration. Using Two-dimensional Difference Gel Electrophoresis, the changes in the proteome of ZSB adapted P. aeruginosa PAO1 were examined by comparison with the non-adapted P. aeruginosa PAO1. The proteome of the adapted P. aeruginosa PAO1 strain differed significantly from the non-adapted in the abundance of two proteins when both strains were grown under stressing conditions. One protein could be identified as the outer membrane protein D that plays a role in uptake of basic amino acids as well as in carbapeneme resistance. The second protein has been identified as alkyl peroxide reductase subunit F. Our data indicated a slight increase in abundance of alkyl peroxide reductase F (AhpF) in the case of ZSB passaged P. aeruginosa PAO1. Higher abundance of Ahp has been discussed in the literature as a promoter of accelerated detoxification of benzene derivatives. The observed up-regulated AhpF thus appears to be connected to an increased tolerance against ZSB. Changes in the abundance of proteins connected to oxidative stress were also found after short-time exposure of P. aeruginosa PAO1 to the ZSB. Furthermore, adapted P. aeruginosa PAO1 showed increased tolerance against hydrogen peroxide and, in addition, showed accelerated degradation of ZSB, as determined by HPLC measurements.     

Antimicrobial Peptide defenses in amphibian skin

One of the most urgent problems in conservation biology todayis the continuing loss of amphibian populations on a globalscale. Recent amphibian population declines in Australia, CentralAmerica, the western United States, Europe, and Africa havebeen linked to a pathogenic chytrid fungus, Batrachochytriumdendrobatidis, which infects the skin. The skin of amphibiansis critical for fluid balance, respiration, and transport ofessential ions; and the immune defense of the skin must be integratedwith these physiological responses. One of the natural defensesof the skin is production of antimicrobial peptides in granularglands. Discharge of the granular glands is initiated by stimulationof sympathetic nerves. To determine whether antimicrobial skinpeptides play a role in protection from invasive pathogens,purified antimicrobial peptides and natural peptide mixturesrecovered from the skin secretions of a number of species havebeen assayed for growth inhibition of the chytrid fungus. Thegeneral findings are that most species tested have one or moreantimicrobial peptides with potent activity against the chytridfungus, and natural mixtures of peptides are also effectiveinhibitors of chytrid growth. This supports the hypothesis thatantimicrobial peptides produced in the skin are an importantdefense against skin pathogens and may affect survival of populations.We also report on initial studies of peptide depletion usingnorepinephrine and the kinetics of peptide recovery followinginduction. Approximately 80 nmoles/g of norepinephrine is requiredto deplete peptides, and peptide stores are not fully recoveredat three weeks following this treatment. Because many specieshave defensive peptides and yet suffer chytrid-associated populationdeclines, it is likely that other factors (temperature, conditionsof hydration, "stress," or pesticides) may alter normal defensesand allow for uncontrolled infection.     

Enzymes of the Tryptophan Synthetic Pathway in Pseudomonas putida

The first four enzymatic activities of the tryptophan synthetic pathway in Pseudomonas putida were found on separate molecules. Gel filtration and density gradient centrifugation experiments did not disclose any associations or aggregations among them. These findings contrast with the situation found in the enteric bacteria, where the first two activities are found in an aggregate and the third and fourth are catalyzed by a single enzyme. Tryptophan synthetase, the last enzyme of the pathway, consists of two dissociable components. The affinity of these components is less in P. putida than is the case in Escherichia coli.     

Transforming kelp into a marine bioreactor

The past decade has seen the genetic engineering of various types of seaweed. To date, genetic transformation studies have been carried out in several seaweeds, including the red seaweeds Porphyra, Gracilaria, Grateloupia, Kappaphycus and Ceramium and the green seaweed Ulva. A genetic transformation model system has been established in the most commonly cultivated seaweed, the brown seaweed Laminaria japonica (kelp), based on the transfer of technology used in land plant transformation and also by modulating the seaweed life cycle. This model showed the potential for application of transgenic kelp to the production of valuable products and an indoor cultivation system for transgenic kelp was proposed, taking into account necessary factors for bio-safety. In this review, the establishment at use of the kelp transformation model is introduced, highlighting the potential for transforming kelp into a marine bioreactor.     

Recombinant Production and Intein-Mediated Purification of an Antimicrobial Peptide,BR2

Nowadays, targeted therapy of cancer is under intensive focus of many investigations due to severe side effects imposed by various cancer chemotherapeutics. BR2 is a modified antimicrobial cell penetrating peptide with confirmed capability of delivering various cargos specifically to cancerous cells. However, because of its small size, its recombinant production by conventional methods is difficult, and its chemical synthesis imposes high cost. Hence, the aim of the present study was to evaluate if recombinant production and intein-mediated purification of this peptide is possible and finally evaluate its safety as a suitable targeted drug delivery vector on cancer and normal cell lines. In this regard, the coding sequence of BR2 was cloned in pTBX1 to be expressed in-frame with GyrA intein and then subjected to inducible protein expression using IPTG. Afterwards, the expressed protein was transferred to chitin-loaded columns and the peptide was purified according to manufacrure’s instruction. SDS–PAGE and Western blot analysis confirmed the expression of BR2-GyrA fusion protein by showing a band of approximately 31 kDa. Moreover, SDS–PAGE of the purified peptide showed a band of approximately 3 kDa, confirming the successful purification of BR2. Finally, in order to evaluate the safety of the produced peptide, its effects was evaluated on MCF-7 and HEK-293 cell lines by MTT assay, and compared to the effects of chemically synthesized BR2. Statistical analysis of the MTT assay results showed that the recombinantly produced peptide had no significant toxic effects on MCF-7 and HEK 293 cells, comparing to negative control, and this was similar to the effects of the synthetic BR2. Hence, the recombinant BR2 can be used for production of novel vehicles for targeted delivery of cytotoxic cargos in the future.     

Enteric YaiW Is a Surface-Exposed Outer Membrane Lipoprotein That Affects Sensitivity to an Antimicrobial Peptide

yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions.     

牛气管黏膜抗菌肽成熟肽基因的克隆及序列分析

目的:获得牛气管黏膜抗菌肽(bTAP)成熟肽的基因序列,为后续的研究工作奠定基础。方法:从新屠宰的黄牛气管黏膜中提取总RNA,反转录获得cDNA,以此cDNA为模板进行PCR扩增目的片段,并将其克隆至pMD18-T载体中,经鉴定随机挑选1个阳性重组子进行测序,将测序结果与已报道的序列进行比较,并做NCBIBlast比对。结果:PCR扩增出bTAP成熟肽基因,核苷酸序列测定验证了其正确性;NCBIBlast比对表明,与bTAP成熟肽基因同源性较高的分别是牛β-防御素11、牛β-防御素12、牛β-防御素402、牛β-防御素403、绵羊β-防御素1、绵羊β-防御素2、山羊β-防御素1及山羊β-防御素2,核苷酸序列同源性分别为78.07%、78.95%、80.70%、83.33%、83.33%、80.70%、81.58%和81.58%,氨基酸序列同源性分别为68.42%、65.79%、68.42%、76.32%、71.05%、63.16%、63.16%和68.42%。结论:成功克隆了bTAP成熟肽的基因序列,NCBIBlas比对表明bTAP与防御素可能来自一个共同的祖系基因。     

Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of “dead-cell stains” (SYTOX orange and SYTOX green) and “live-cell stains” (DRAQ5 and SYTO 61) and also 4′,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.     

Serum Stability and Physicochemical Characterization of a Novel Amphipathic Peptide C6M1 for SiRNA Delivery

The efficient delivery of nucleic acids as therapeutic agents is a major challenge in gene therapy. Peptides have recently emerged as a novel carrier for delivery of drugs and genes. C6M1 is a designed amphipathic peptide with the ability to form stable complexes with short interfering RNA (siRNA). The peptide showed a combination of random coil and helical structure in water but mainly adopted a helical conformation in the presence of anions or siRNA. Revealed by dynamic light scattering (DLS) and microscopy techniques, the interaction of C6M1 and siRNA in water and HEPES led to complexes of ∼70 and ∼155 nm in size, respectively, but showed aggregates as large as ∼500 nm in PBS. The time-dependent aggregation of the complex in PBS was studied by DLS and fluorescence spectroscopy. At molar ratio of 15∶1, C6M1 was able to completely encapsulate siRNA; however, higher molar ratios were required to obtain stable complexes. Naked siRNA was completely degraded in 4 h in the solution of 50% serum; however C6M1 protected siRNA against serum RNase over the period of 24 h. Western blotting experiment showed ∼72% decrease in GAPDH protein level of the cells treated with C6M1-siRNA complexes while no significant knockdown was observed for the cells treated with naked siRNA.     

Increasing stability and toxicity of Pseudomonas exotoxin by attaching an antiproteasic Peptide

Trypsin-like activities are present within the endocytic pathway and allow cells to inactivate a fraction of incoming toxins, such as Pseudomonas exotoxin (PE), that require endocytic uptake before reaching the cytosol to inactivate protein synthesis. PE is a favorite toxin for building immunotoxins. The latter are promising molecules to fight cancer or transplant rejection, and producing more active toxins is a key challenge. More broadly, increasing protein stability is a potentially useful approach to improve the efficiency of therapeutic proteins. We report here that fusing an antiproteasic peptide (bovine pancreatic trypsin inhibitor, BPTI) to PE increases its toxicity to human cancer cell lines by 20-40-fold. Confocal microscopic examination of toxin endocytosis, digestion, and immunoprecipitation experiments showed that the fused antiproteasic peptide specifically protects PE from trypsin-like activities. Hence, the attached BPTI acts as a bodyguard for the toxin within the endocytic pathway. Moreover, it increased the PE elimination half-time in mice by 70%, indicating that the fused BPTI stabilizes the toxin in vivo. This BPTI-fusion approach may be useful for protecting other circulating or internalized proteins of therapeutic interest from premature degradation.     

Evidence For Autogenous Regulation of Pseudomonas putida Tryptophan Synthase

Studies of a trpA mutant constitutive for tryptophan synthase production support the hypothesis of autogenous regulation (R. F. Goldberger, 1974; A. R. Proctor and I. P. Crawford, 1975) of the Pseudomonas putida trpAB loci.     

海洋侧孢短芽孢杆菌Brevibacillus laterosporus Lh-1 产抗菌肽R-1的培养条件优化

采用Plackett-Burman(PB)设计和响应面分析(RSM)方法, 对一株海洋侧孢短芽孢杆菌Lh-1产抗菌肽R-1的发酵条件进行了优化。研究中数据的统计和分析均使用MINITAB 15.0。在研究中, 首先使用了Plackett-Burman (PB)设计对影响Lh-1产抗菌肽的15个因素进行了筛选, 得到了影响产量的显著因素为：葡萄糖、蛋白胨和氯化钙。在此基础上采用响应面法对该3个显著因素的最佳水平范围进行研究, 得到的最佳浓度为 15.72 g/L 葡萄糖、6.01 g/L 蛋白胨和3.29     

Antimicrobial resistance of Pseudomonas aeruginosa biofilms

Resistance to antimicrobial agents is the most important feature of biofilm infections. As a result, infections caused by bacterial biofilms are persistent and very difficult to eradicate. Although several mechanisms have been postulated to explain reduced susceptibility to antimicrobials in bacterial biofilms, it is becoming evident that biofilm resistance is multifactorial. The contribution of each of the different mechanisms involved in biofilm resistance is now beginning to emerge.