Trypanosoma cruzi: Synergistic cytotoxicity of multiple amphipathic anti-microbial peptides to T. cruzi and potential bacterial hosts

The parasite Trypanasoma cruzi is responsible for Chagas disease and its triatomine vector, Rhodnius prolixus, has a symbiotic relationship with the soil bacterium, Rhodococcus rhodnii.R. rhodnii that was previously genetically engineered to produce the anti-microbial peptide, cecropin A was co-infected with T. cruzi into R. prolixus resulting in clearance of the infectious T. cruzi in 65% of the vectors. Similar anti-microbial peptides have been isolated elsewhere and were studied for differential toxicity against T. cruzi and R. rhodnii. Of the six anti-microbial peptides tested, apidaecin, magainin II, melittin, and cecropin A were deemed potential candidates for the Chagas paratransgenic system as they were capable of killing T.cruzi at concentrations that exhibit little or no toxic effects on R. rhodnii. Subsequent treatments of T. cruzi with these peptides in pair-wise combinations resulted in synergistic killing, indicating that improvement of the 65% parasite clearance seen in previous experiments may be possible utilizing combinations of different anti-microbial peptides.     

Insect juvenile hormone binding protein shows ancestral fold present in human lipid-binding proteins

Low molecular weight juvenile hormone binding proteins (JHBPs) are specific carriers of juvenile hormone (JH) in the hemolymph of butterflies and moths. As hormonal signal transmitters, these proteins exert a profound effect on insect development. The crystal structure of JHBP from Galleria mellonella shows an unusual fold consisting of a long α-helix wrapped in a highly curved antiparallel β-sheet. JHBP structurally resembles the folding pattern found in tandem repeats in some mammalian lipid-binding proteins, with similar organization of one cavity and a disulfide bond between the long helix and the β-sheet. JHBP reveals, therefore, an archetypal fold used by nature for hydrophobic ligand binding. The JHBP molecule possesses two hydrophobic cavities. Several lines of experimental evidence conclusively indicate that JHBP binds JH in only one cavity, close to the N- and C-termini, and that this binding induces a structural change. The second cavity, located at the opposite end of the molecule, could bind another ligand.     

Plant protection in sustainable systems of agriculture

Abstract

A comprehensive evaluation of 13 plant essential oils namely: caraway, chenopodium, cinnamon, clove, eucalyptus, garlic, geranium, lemon, matrecary, peppermint, rose, rosemary and thyme and 14 of their monoterpenoidal constituents: borneol, camphor, carvacrol, carveol, carvone (R and S), chlorothymol, cineol, cinnamaldehyde, citronellol, eugenol, geraneol, menthol, and thymol was investigated for their antibacterial activity against the two phytopathogenic bacteria, Agrobacterium tumefaciens and Erwinia carotovora var carotovora. Trials for improving the antibacterial activity by either structure modification or mixing with the three synergists: piperonyl butoxide, Triton X-100 and Tween 20 were achieved. New N-methyl carbamate derivatives based on monoterpenoids were introduced. Cinnamon, clove, chenopodium, caraway, rosemary and thyme oils proved to possess good antibacterial activity. Thymol, chlorothymol, and carvacrol were potent against the tested bacteria and quite close to the effect of the standard. Conversion of menthol into its carbamate derivative gave good enhancement in the bactericidal activity at all concentrations compared to menthol itself. Also, the carbamate derivatives of β-citronellol and borneol improved the bactericidal activity against E. carotovora, but decreased it against A. tumefaciens. Synergistic antibacterial activity of the tested monoterpenoids was observed when combined with each of the three synergists particularly with Triton X-100.     

Novel fungicidal benzylsulfanyl-phenylguanidines

A series of substituted benzylsulfanyl-phenylamines was synthesized, of which four substituted benzylsulfanyl-phenylguanidines (665, 666, 667 and 684) showed potent fungicidal activity (minimal fungicidal concentration, MFC ? 10 μM for Candida albicans and Candida glabrata). A benzylsulfanyl-phenyl scaffold with an unsubstituted guanidine resulted in less active compounds (MFC = 50-100 μM), whereas substitution with an unsubstituted amine group resulted in compounds without fungicidal activity. Compounds 665, 666, 667 and 684 also showed activity against single C. albicans biofilms and biofilms consisting of C. albicans and Staphylococcus epidermidis (minimal concentration resulting in 50% eradication of the biofilm, BEC50 ? 121 μM for both biofilm setups). Compounds 665 and 666 combined potent fungicidal (MFC = 5 μM) and bactericidal activity (minimal bactericidal concentration, MBC for S. epidermidis ? 4 μM). In an in vivo Caenorhabditis elegans model, compounds 665 and 667 exhibited less toxicity than 666 and 684. Moreover, addition of those compounds to Candida-infected C. elegans cultures resulted in increased survival of Candida-infected worms, demonstrating their in vivo efficacy in a mini-host model.     

An ATPase inhibitory peptide with antibacterial and ion current effects

An 84-residue bactericidal peptide, PSK, was purified from a Chrysomya megacephala fly larvae preparation. Its amino acid sequence is similar to that of a previously reported larval peptide of the Drosophila genus (SK84) noticed for its anticancer and antimicrobial properties. The PSK sequence is also homologous to mitochondrial ATPase inhibitors from insects to humans (35–65% sequence identity), indicating an intracellular protein target and possible mechanism for PSK. It contains a cluster of six glycine residues, and has several two- and three-residue repeats. It is active against both Gram-positive and Gram-negative bacteria via a mechanism apparently involving cell membrane disintegration and inhibition of ATP hydrolysis. In addition, PSK induces an inward cationic current in pancreatic β cells. Together, the findings identify a bioactive peptide of the ATPase inhibitor family with specific effects on both prokaryotic and mammalian cells.     

核糖核酸酶5在宿主防御中的作用及机制

抗菌肽是机体天然免疫的重要组成部分。核糖核酸酶5（ribonuclease5,RNASE5;又名angiogenin）属于核糖核酸酶A超家族,是一个分泌型小分子蛋白质,广泛分布于机体需要抵御外界病原微生物的组织中。RNASE5对病毒、细菌以及真菌都存在抑制效应,具有广谱抗菌特点,但其表达和活性受到宿主生理状态和外界环境多层次的调控。RNASE5存在多样的抗微生物作用机制,其带正电荷的理化特性破坏微生物细胞壁,而其核糖核酸酶活性则是杀伤真菌所必须的。除了直接作用于微生物外,RNASE5还可作为重要因子调节宿主免疫功能,参与多种病理过程。本文综述了RANSE5的结构、生物活性与功能、作用特点与机制,并讨论了在其研究中存在的问题,以期为今后的研究提供新思路和新方向。     

Damage of Escherichia coli membrane by bactericidal agent polyhexamethylene guanidine hydrochloride: micrographic evidences

Aims: The purpose of this study was to provide micrographic evidences for the damaged membrane structure and intracellular structure change of Escherichia coli strain 8099, induced by polyhexamethylene guanidine hydrochloride (PHMG). Methods and Results: The bactericidal effect of PHMG on E. coli was investigated based on β‐galactosidase activity assay, fluorescein‐5‐isothiocyanate confocal laser scanning microscopy, field emission scanning electron microscopy and transmission electron microscopy. The results revealed that a low dose (13 μg ml^?1) of PHMG slightly damaged the outer membrane structure of the treated bacteria and increased the permeability of the cytoplasmic membrane, while no significant damage was observed to the morphological structure of the cells. A high dose (23 μg ml^?1) of PHMG collapsed the outer membrane structure, led to the formation of a local membrane pore across the membrane and badly damaged the internal structure of the cells. Subsequently, intracellular components were leaked followed by cell inactivation. Conclusions: Dose‐dependent membrane disruption was the main bactericidal mechanism of PHMG. The formation of the local membrane pores was probable after exposure to a high dose (23 μg ml^?1) of PHMG. Micrographic evidences were provided about the damaged membrane structure and intracellular structure change of E. coli. Significance and Impact of the Study: The presented information helps understand the bactericidal mechanism of PHMG by membrane damage.     

Bactericidal activity and oral pathogen inactivation by electromagnetic wave irradiation

Aims: The aim of this work was to clarify the effects of electromagnetic wave irradiation (EMWI) on oral bacterial pathogens. Methods and Results: A Gram‐negative (Porphyromonas gingivalis) or Gram‐positive (Streptococcus mutans, S. intermedius, Enterococcus faecalis) bacterial suspension was irradiated by EMW apparatus (500–1000 kHz, 5–15 times, 1 s time^?1). Quantification of survival bacteria by CFU counting revealed that EMWI exhibited marked bactericidal activity against all tested bacteria and bactericidal activity at 500 kHz increased in an irradiation number‐dependent manner. After EMWI at 500 kHz, scanning electron microscopic observations showed that the chain of S. mutans cells was shortened after 5 irradiations and the outlines of bacterial cells (S. mutans and P. gingivalis) were unclear after 5–10 irradiations. EMWI inhibited the inductive effect of S. mutans on pro‐inflammatory cytokine production in human monocytes and this inhibitory effect was comparable with that of heat‐killed bacteria. Furthermore, using an enzyme activity assay, EMWI partially inactivated the activities of gingipains from P. gingivalis. Conclusions: These findings demonstrated that EMWI has inactivation and bactericidal activities against single microbial species among four kinds of oral pathogens. Significance and Impact of the Study: Electromagnetic wave irradiation may be applicable for medical disinfection and sterilization, such as refractory periapical periodontitis.     

Lysine substitutions convert a bacterial-agglutinating peptide into a bactericidal peptide that retains anti-lipopolysaccharide activity and low hemolytic activity

GL13NH2 is a bacteria-agglutinating peptide derived from the sequence of the salivary protein parotid secretory protein (PSP, BPIFA2, SPLUNC2, C20orf70). The peptide agglutinates both Gram negative and Gram positive bacteria, and shows anti-lipopolysaccharide activity in vitro and in vivo. However, GL13NH2 does not exhibit bactericidal activity. To generate a more cationic peptide with potential bactericidal activity, three amino acid residues were replaced with lysine residues to generate the peptide GL13K. In this report, the antibacterial and anti-inflammatory activities of GL13K were characterized. GL13K had lost the ability to agglutinate bacteria but gained bactericidal activity. Substitution of individual amino acids in GL13K with alanine did not restore bacterial agglutination. GL13K was bactericidal against Pseudomonas aeruginosa, Streptococcus gordonii and Escherichia coli but not Porphyromonas gingivalis. Unlike the agglutinating activity of GL13NH2, the bactericidal activity of GL13K against P. aeruginosa was retained in the presence of saliva. Both GL13NH2 and GL13K exhibited anti-lipopolysaccharide activity. In GL13K, this activity appeared to depend on a serine hydroxyl group. GL13K protected mice from lipopolysaccharide-induced sepsis and the peptide exhibited a low level of hemolysis, suggesting that it may be suitable for in vivo application.     

Effects of dimerization of the cell‐penetrating peptide Tat analog on antimicrobial activity and mechanism of bactericidal action

The cell‐penetrating peptide Tat (48–60) (GRKKRRQRRRPPQ) derived from HIV‐1 Tat protein showed potent antibacterial activity (MIC: 2–8 µM ). To investigate the effect of dimerization of Tat (48–60) analog, [Tat(W): GRKKRRQRRRPWQ‐NH₂], on antimicrobial activity and mechanism of bactericidal action, its dimeric peptides, di‐Tat(W)‐C and di‐Tat(W)‐K, were synthesized by a disulfide bond linkage and lysine linkage of monomeric Tat(W), respectively. From the viewpoint of a weight basis and the monomer concentration, these dimeric peptides displayed almost similar antimicrobial activity against six bacterial strains tested but acted more rapidly against Staphylococcus aureus on kinetics of bactericidal activity, compared with monomeric Tat(W). Unlike monomeric Tat(W), these dimeric peptides significantly depolarized the cytoplasmic membrane of intact S. aureus cells at MIC and induced dye leakage from bacterial‐membrane‐mimicking egg yolk L ‐α‐phosphatidylethanolamine/egg yolk L ‐α‐phosphatidyl‐DL ‐glycerol (7:3, w/w) vesicles. Furthermore, these dimeric peptides were less effective to translocate across lipid bilayers than monomeric Tat(W). These results indicated that the dimerization of Tat analog induces a partial change in the mode of its bactericidal action from intracellular target mechanism to membrane‐targeting mechanism. Collectively, our designed dimeric Tat peptides with high antimicrobial activity and rapid bactericidal activity appear to be excellent candidates for future development as novel antimicrobial agents. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.