Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli

Antimicrobial peptides are produced by all organisms in response to microbial invasion and are considered as promising candidates for future antibiotics. There is a wealth of evidence that many of them interact and increase the permeability of bacterial membranes as part of their killing mechanism. However, it is not clear whether this is the lethal step. To address this issue, we studied the interaction of the antimicrobial peptide temporin L with Escherichia coli by using fluorescence, confocal and electron microscopy. The peptide previously isolated from skin secretions of the frog Rana temporaria has the sequence FVQWFSKFLGRIL-NH2. With regard to fluorescence microscopy, we applied, for the first time, a triple-staining method based on the fluorochromes 5-cyano-2,3-ditolyl tetrazolium chloride, 4´,6-diamidino-2-phenylindole and FITC. This technique enabled us to identify, in the same sample, both living and total cells, as well as bacteria with altered membrane permeability. These results reveal that temporin L increases the permeability of the bacterial inner membrane in a dose-dependent manner without destroying the cell's integrity. At low peptide concentrations, the inner membrane becomes permeable to small molecules but does not allow the killing of bacteria. However, at high peptide concentrations, larger molecules, but not DNA, leak out, which results in cell death. Very interestingly, in contrast with many antimicrobial peptides, temporin L does not lyse E. coli cells but rather forms ghost-like bacteria, as observed by scanning and transmission electron microscopy. Besides shedding light on the mode of action of temporin L and possibly that of other antimicrobial peptides, the present study demonstrates the advantage of using the triple-fluorescence approach combined with microscopical techniques to explore the mechanism of membrane-active peptides in general.

Download Full-text

Long-term effects of the proline-rich antimicrobial peptide Oncocin112 on the Escherichia coli translation machinery

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013587 ◽

2020 ◽

Vol 295 (38) ◽

pp. 13314-13325

Author(s):

Yanyu Zhu ◽

James C. Weisshaar ◽

Mainak Mustafi

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptides ◽

Binding Site ◽

Elongation Factor ◽

Microscopy Study ◽

Single Step ◽

Long Term Effects ◽

Bacterial Membranes

Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro. Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of ∼20 aa/s.

Download Full-text

Proteomic Adaptation of Streptococcus pneumoniae to the Antimicrobial Peptide Human Beta Defensin 3 (hBD3) in Comparison to Other Cell Surface Stresses

Microorganisms ◽

10.3390/microorganisms8111697 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1697

Author(s):

Pierre-Alexander Mücke ◽

Anne Ostrzinski ◽

Sven Hammerschmidt ◽

Sandra Maaß ◽

Dörte Becher

Keyword(s):

Streptococcus Pneumoniae ◽

Cell Surface ◽

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Specific Cell ◽

Similar Response ◽

Respiratory Pathogens ◽

Human Respiratory Tract ◽

Bacterial Membranes ◽

Surface Stresses

The antimicrobial peptide human Beta defensin 3 (hBD3) is an essential part of the innate immune system and is involved in protection against respiratory pathogens by specifically permeabilizing bacterial membranes. The Gram-positive bacterium Streptococcus pneumoniae causes serious diseases including pneumonia, meningitis, and septicemia, despite being frequently exposed to human defense molecules, including hBD3 during colonization and infection. Thus, the question arises how pneumococci adapt to stress caused by antimicrobial peptides. We addressed this subject by analyzing the proteome of S. pneumoniae after treatment with hBD3 and compared our data with the proteomic changes induced by LL-37, another crucial antimicrobial peptide present in the human respiratory tract. As antimicrobial peptides usually cause membrane perturbations, the response to the membrane active cationic detergent cetyltrimethylammonium bromide (CTAB) was examined to assess the specificity of the pneumococcal response to antimicrobial peptides. In brief, hBD3 and LL-37 induce a similar response in pneumococci and especially, changes in proteins with annotated transporter and virulence function have been identified. However, LL-37 causes changes in the abundance of cell surface modification proteins that cannot be observed after treatment with hBD3. Interestingly, CTAB induces unique proteomic changes in S. pneumoniae. Though, the detergent seems to activate a two-component system that is also activated in response to antimicrobial peptide stress (TCS 05). Overall, our data represent a novel resource on pneumococcal adaptation to specific cell surface stresses on a functional level. This knowledge can potentially be used to develop strategies to circumvent pneumococcal resistance to antimicrobial peptides.

Download Full-text

Induction of the Cpx Envelope Stress Pathway Contributes to Escherichia coli Tolerance to Antimicrobial Peptides

Applied and Environmental Microbiology ◽

10.1128/aem.02593-13 ◽

2013 ◽

Vol 79 (24) ◽

pp. 7770-7779 ◽

Cited By ~ 21

Author(s):

Bianca Audrain ◽

Lionel Ferrières ◽

Amira Zairi ◽

Guillaume Soubigou ◽

Curtis Dobson ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Polymyxin B ◽

Genetic Response ◽

Content Type ◽

E Coli ◽

Multiresistant Bacteria ◽

Envelope Stress ◽

Stress Pathway

ABSTRACTAntimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response ofEscherichia coliupon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σEenvelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S4dermaseptin, also activate severalE. colienvelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes toE. colitolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show thatE. colisenses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway toE. colitolerance to antimicrobial peptides.

Download Full-text

Heterologous Expression and Purification of the Antimicrobial Peptide Buforin II

Bulletin of Medical Sciences ◽

10.2478/orvtudert-2019-0010 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Boda Ferenc-András ◽

Szabó Zoltán-István ◽

Szőcs Erika ◽

Salamon Pál ◽

Orbán Csongor ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Heterologous Expression ◽

Antimicrobial Peptide ◽

Solid Phase ◽

Host Cells ◽

Fusion Partner ◽

Monitoring And Control ◽

Expression And Purification ◽

Recombinant Peptide ◽

Bacterial Membranes

AbstractAntimicrobial peptides are natural substances that have played a role in the development of the adaptive immune system, and are currently involved in the prevention of infections, through their direct antimicrobial and immunomodulatory properties. While the amino acid composition and spatial structure vary, most antibacterial peptides have a positive surface charge, which allows them to bind to the negative bacterial membranes. Buforin II is a widely studied antimicrobial peptide first obtained through the structural modification of buforin I, a peptide isolated from Bufo gargarizans. The peptide showed significant antibacterial activity against Gram-positive and Gram-negative bacterial strains. The mechanism of action of buforin II differs from that of other antimicrobial peptides, as it binds directly to bacterial DNA and RNA. The aim of our study was to obtain recombinant buforin II with a ubiquitin fusion partner, through heterologous expression in Escherichia coli Rosetta™ (DE3)pLysS cells, using a laboratory scale biore-actor. The incubation of expression host cells in a bioreactor allowed the constant monitoring and control of the process parameters, leading to high biomass levels and an increased production rate of the peptide. The parameters used during incubation were: 37°C, pH=6.9 and dissolved oxygen level above 40%. Purification of the recombinant protein was accomplished by affinity chromatography using a Ni-chelate solid phase to which the 10xHistag of our construct showed affinity. Method optimisation consisted in the use of gradient and linear elution, of which the latter was found to be more effective. Digestion of the fusion partner from the target peptide was performed with ubiquitin carboxyl-terminal hydrolase enzyme. The expression and purification protocols developed in our experiment allow the production of a significant amount of buforin II, allowing its use for further research. Furthermore, the presented methods could be suitable for industrial production of the recombinant peptide..

Download Full-text

Damage of the Bacterial Cell Envelope by Antimicrobial Peptides Gramicidin S and PGLa as Revealed by Transmission and Scanning Electron Microscopy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00124-10 ◽

2010 ◽

Vol 54 (8) ◽

pp. 3132-3142 ◽

Cited By ~ 250

Author(s):

Mareike Hartmann ◽

Marina Berditsch ◽

Jacques Hawecker ◽

Mohammad Fotouhi Ardakani ◽

Dagmar Gerthsen ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Antimicrobial Peptides ◽

Lipid Membrane ◽

Ultrastructural Changes ◽

Morphological Alterations ◽

Bacterial Membranes ◽

E Coli ◽

Gramicidin S ◽

Scanning Electron

ABSTRACT Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the ultrastructural changes in bacteria induced by antimicrobial peptides (AMPs). Both the β-stranded gramicidin S and the α-helical peptidyl-glycylleucine-carboxyamide (PGLa) are cationic amphiphilic AMPs known to interact with bacterial membranes. One representative Gram-negative strain, Escherichia coli ATCC 25922, and one representative Gram-positive strain, Staphylococcus aureus ATCC 25923, were exposed to the AMPs at sub-MICs and supra-MICs in salt-free medium. SEM revealed a shortening and swelling of the E. coli cells, and multiple blisters and bubbles formed on their surface. The S. aureus cells seemed to burst upon AMP exposure, showing open holes and deep craters in their envelope. TEM revealed the formation of intracellular membranous structures in both strains, which is attributed to a lateral expansion of the lipid membrane upon peptide insertion. Also, some morphological alterations in the DNA region were detected for S. aureus. After E. coli was incubated with AMPs in medium with low ionic strength, the cells appeared highly turgid compared to untreated controls. This observation suggests that the AMPs enhance osmosis through the inner membrane, before they eventually cause excessive leakage of the cellular contents. The adverse effect on the osmoregulatory capacity of the bacteria is attributed to the membrane-permeabilizing action of the amphiphilic peptides, even at low (sub-MIC) AMP concentrations. Altogether, the results demonstrate that both TEM and SEM, as well as appropriate sample preparation protocols, are needed to obtain detailed mechanistic insights into peptide function.

Download Full-text

Investigation into Formation of Lipid Hydroperoxides from Membrane Lipids in Escherichia coli O157:H7 following Exposure to Hot Water

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-394 ◽

2015 ◽

Vol 78 (6) ◽

pp. 1197-1202 ◽

Cited By ~ 1

Author(s):

THELMA F. CÁLIX-LARA ◽

KATIE R. KIRSCH ◽

MARGARET D. HARDIN ◽

ALEJANDRO CASTILLO ◽

STEPHEN B. SMITH ◽

...

Keyword(s):

Escherichia Coli ◽

Lactic Acid ◽

Membrane Lipids ◽

Lipid Hydroperoxide ◽

Hot Water ◽

Lipid Hydroperoxides ◽

Escherichia Coli O157 ◽

Dependent Manner ◽

Bacterial Membranes ◽

E Coli

Although studies have shown antimicrobial treatments consisting of hot water sprays alone or paired with lactic acid rinses are effective for reducing Escherichia coli O157:H7 loads on beef carcass surfaces, the mechanisms by which these interventions inactivate bacterial pathogens are still poorly understood. It was hypothesized that E. coli O157:H7 exposure to hot water in vitro at rising temperatures for longer time periods would result in increasing deterioration of bacterial outer membrane lipids, sensitizing the pathogen to subsequent lactic acid application. Cocktails of E. coli O157:H7 strains were subjected to hot water at 25 (control) 65, 75, or 85°C incrementally up to 60 s, after which surviving cells were enumerated by plating. Formation of lipid hydroperoxides from bacterial membranes and cytoplasmic accumulation of l-lactic acid was quantified spectrophotometrically. Inactivation of E. coli O157:H7 proceeded in a hot water exposure duration- and temperature-dependent manner, with populations being reduced to nondetectable numbers following heating of cells in 85°C water for 30 and 60 s (P < 0.05). Lipid hydroperoxide formation was not observed to be dependent upon increasing water temperature or exposure period. The data suggest that hot water application prior to organic acid application may function to increase the sensitivity of E. coli O157:H7 cells by degrading membrane lipids.

Download Full-text

Lentivirus Lytic Peptide 1 Perturbs both Outer and Inner Membranes of Serratia marcescens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.6.2041-2045.2002 ◽

2002 ◽

Vol 46 (6) ◽

pp. 2041-2045 ◽

Cited By ~ 25

Author(s):

Shruti M. Phadke ◽

Vanja Lazarevic ◽

Caroline C. Bahr ◽

Kazi Islam ◽

Donna Beer Stolz ◽

...

Keyword(s):

Electron Microscopy ◽

Antimicrobial Peptides ◽

Serratia Marcescens ◽

Outer Membrane ◽

Polymyxin B ◽

Mechanisms Of Action ◽

Lytic Peptide ◽

Bacterial Membranes ◽

Lentivirus Lytic Peptide

ABSTRACT Bis-lentivirus lytic protein 1 (Bis-LLP1) and polymyxin B exhibited similar killing activities against Serratia marcescens. By electron microscopy, bis-LLP1 interacted with the outer and cytoplasmic bacterial membranes, while polymyxin B affected only the outer membrane. The results of standard biochemical probes supported the findings of the electron microscopy studies, suggesting that these antimicrobial peptides have different mechanisms of action.

Download Full-text

The effects of organic pesticides on inner membrane permeability in Escherichia coli ML35

Cell Biology and Toxicology ◽

10.1007/s10565-005-0123-4 ◽

2005 ◽

Vol 21 (2) ◽

pp. 73-81 ◽

Cited By ~ 8

Author(s):

K. Guven ◽

M. Yolcu ◽

R. Gul-Guven ◽

S. Erdogan ◽

D. De Pomerai

Keyword(s):

Escherichia Coli ◽

Membrane Permeability ◽

Inner Membrane ◽

Organic Pesticides

Download Full-text

Antimicrobial peptide bsn-37 inhibits the growth of Escherichia coli by targeting its cell wall and membrane

Materials Express ◽

10.1166/mex.2020.1773 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1260-1264

Author(s):

Jingyu Fu ◽

Hao Yang ◽

Hongliang Wang ◽

Jun Ke ◽

Debao Kong

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Antimicrobial Peptides ◽

Nucleic Acids ◽

Antimicrobial Peptide ◽

Mechanism Of Action ◽

E Coli ◽

Colony Counting ◽

Kinetics Of ◽

Growth And Reproduction

To understand the mechanism of action of the antimicrobial peptide bsn-37 on Escherichia coli (E. coli), we investigated its effects on leakage of ultraviolet-absorbing substances, proteins, and nucleic acids from E. coli CVCC1568. The bacteriostatic kinetics of antimicrobial peptides was determined by colony counting. Our study showed that bsn-37 could effectively inhibit the growth and reproduction of E. coli by disrupting its cell wall and membrane.

Download Full-text