Fluorescence and Electron Microscopy Methods for Exploring Antimicrobial Peptides Mode(s) of Action

Marcellini, L; Giammatteo, M.; Aimola, Pierpaolo; Mangoni, Maria Luisa

doi:10.1007/978-1-60761-594-1_16

Cited by 17 publications

(15 citation statements)

References 10 publications

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“…The bacteria were exposed to a low (0.5 mM), medium (1 mM) and high (2 mM) concentrations of MGO in the same way as for the antibacterial assay. For SEM a 100 µl volume of the bacteria was transferred to the wells of a 24-well plate containing poly-L-lysine coated cover glass slides [13]. After 90 min incubation at 30°C, samples were fixed for 1 h using a solution of 2.5 % formaldehyde and gluteraldehyde in 0.075 M sodium potassium phosphate (NaP) buffer pH 7.4.…”

Section: Scanning Electron Microscopymentioning

confidence: 99%

How methylglyoxal kills bacteria: An ultrastructural study

Rabie

Serem

Oberholzer

et al. 2016

Ultrastructural Pathology

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Antibacterial activity of honey is due to the presence of methylglyoxal (MGO), H 2 O 2 , bee defensin as well as polyphenols. High MGO levels in manuka honey are the main source of antibacterial activity. Manuka honey has been reported to reduce the swarming and swimming motility of Pseudomonas aeruginosa due to de-flagellation. Due to the complexity of honey it is unknown if this effect is directly due to MGO. In this ultrastructural investigation the effects of MGO on the morphology of bacteria and specifically the structure of fimbriae and flagella were and E. coli morphology was then evaluated. At 0.5 mM MGO, bacteria structure was unaltered.For both bacteria at 1 mM MGO fewer fimbriae were present and the flagella were less or absent. Identified structures appeared stunted and fragile. At 2 mM MGO fimbriae and flagella were absent while the bacteria were rounded with shrinkage and loss of membrane integrity.Antibacterial MGO causes alterations in the structure of bacterial fimbriae and flagella which would limit bacteria adherence and motility.

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Section: Scanning Electron Microscopymentioning

confidence: 99%

How methylglyoxal kills bacteria: An ultrastructural study

Rabie

Serem

Oberholzer

et al. 2016

Ultrastructural Pathology

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“…The method for negativestained ultrathin sections was adapted from that reported by Marcellini et al (25). Cells were grown as for the growth kinetic experiments (OD 600 , 0.1) in MOPS minimal medium.…”

Section: Bacteriamentioning

confidence: 99%

Deciphering the Mode of Action of the Synthetic Antimicrobial Peptide Bac8c

Spindler

Hale

Giddings

et al. 2011

Antimicrob Agents Chemother

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Bac8c (RIWVIWRR-NH 2 ) is an 8-amino-acid peptide derived from Bac2A (RLARIVVIRVAR-NH 2 ), a C3A/ C11A variant of the naturally occurring bovine peptide, bactenecin (also known as bovine dodecapeptide), the smallest peptide with activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. The effects of Bac8c on Escherichia coli were examined by studying its bacteriostatic and bactericidal properties, demonstrating its effects on proton motive force generation, and visually analyzing (via transmission electron microscopy) its effects on cells at different concentrations, in order to probe the complexities of the mechanism of action of Bac8c. Results were consistent with a two-stage model for the Bac8c mode of action. At sublethal concentrations (3 g/ml), Bac8c addition resulted in transient membrane destabilization and metabolic imbalances, which appeared to be linked to inhibition of respiratory function. Although sublethal concentrations resulted in deleterious downstream events, such as methylglyoxal formation and free radical generation, native E. coli defense systems were sufficient for full recovery within 2 h. In contrast, at the minimal bactericidal concentration (6 g/ml), Bac8c substantially but incompletely depolarized the cytoplasmic membrane within 5 min and disrupted electron transport, which in turn resulted in partial membrane permeabilization and cell death.

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“…The cover slips were then treated with anhydrous ethanol for 30 min and immediately dried over an alcohol lamp. After incubating in 0.1 mg/ml poly (l-lysine) for 2 h, the treated cover slips were washed three times with distilled water and dried at room temperature for at least 3 days before use (33). Individual insects were held on ice in a glass dish, a leg was removed, and a drop of hemolymph was collected from the leg wound (34).…”

Section: Methodsmentioning

confidence: 99%

Proteomic Analysis of Interaction between a Plant Virus and Its Vector Insect Reveals New Functions of Hemipteran Cuticular Protein

Liu

Gray

Huo

et al. 2015

Molecular & Cellular Proteomics

101

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Numerous viruses can be transmitted by their corresponding vector insects; however, the molecular mechanisms enabling virus transmission by vector insects have been poorly understood, especially the identity of vector components interacting with the virus. Here, we used the yeast two-hybrid system to study proteomic interactions of a plant virus (Rice stripe virus, RSV, genus Tenuivirus) with its vector insect, small brown planthopper (Laodelphax striatellus). Sixty-six proteins of L. striatellus that interacted with the nucleocapsid protein (pc3) of RSV were identified. A virus-insect interaction network, constructed for pc3 and 29 protein homologs of Drosophila melanogaster, suggested that nine proteins might directly interact with pc3. Of the 66 proteins, five (atlasin, a novel cuticular protein, jagunal, NAC domain protein, and vitellogenin) were most likely to be involved in viral movement, replication, and transovarial transmission. This work also provides evidence that the novel cuticular protein, CPR1, from L. striatellus is essential for RSV transmission by its vector insect. CPR1 binds the nucleocapsid protein (pc3) of RSV both in vivo and in vitro and colocalizes with RSV in the hemocytes of L. striatellus. Knockdown of CPR1 transcription using RNA interference resulted in a decrease in the concentration of RSV in the hemolymph, salivary glands and in viral transmission efficiency. These data suggest that CPR1 binds RSV in the insect and stabilizes the viral concentration in the hemolymph, perhaps to protect the virus or to help move the virus to the salivary tissues. Our studies provide direct experimental evidence that viruses can use existing vector proteins to aid their survival in the hemolymph. Identifying these pu-

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Fluorescence and Electron Microscopy Methods for Exploring Antimicrobial Peptides Mode(s) of Action

Cited by 17 publications

References 10 publications

How methylglyoxal kills bacteria: An ultrastructural study

How methylglyoxal kills bacteria: An ultrastructural study

Deciphering the Mode of Action of the Synthetic Antimicrobial Peptide Bac8c

Proteomic Analysis of Interaction between a Plant Virus and Its Vector Insect Reveals New Functions of Hemipteran Cuticular Protein

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