August 2023

Antimicrobial Peptide Mastoparan-AF Kills Multi-Antibiotic Resistant Escherichia coli O157:H7 via Multiple Membrane Disruption Patterns and Likely by Adopting 3–11 Amphipathic Helices to Favor Membrane Interaction

The emergence of multiple antibiotic-resistant bacteria, notably, pan-resistant Gram-negative pathogens, which are equipped with an outer membrane barrier of low permeability to antibiotics, has become an important challenge in recent decades following the overuse of antibiotics in humans and animals. *

In particular, the foodborne enteric pathogen Escherichia coli O157:H7 has caused severe or deadly illness cases worldwide. *

Among E. coli O157 isolates, serotype O157:H7 is the most common enteric pathogen isolated from patients with bloody diarrhea and it is also frequently found in non-bloody diarrhea samples. Many of its clinical isolates from humans and animals as well as isolates from contaminated food have been found to develop resistance to several antibiotics. *

Following the first isolation of mastoparan, the most abundant peptide in the hornet or wasp venom, from Vespula lewisii, many homologs of mastoparan were isolated from various hornets and solitary wasps. *

Mastoparan homologs are cationic tetradecapeptides with membrane permeabilizing activity and antimicrobial activity on various bacteria, mast cell degranulation activity, and hemolytic activity. *

In the article “Antimicrobial Peptide Mastoparan-AF Kills Multi-Antibiotic Resistant Escherichia coli O157:H7 via Multiple Membrane Disruption Patterns and Likely by Adopting 3–11 Amphipathic Helices to Favor Membrane Interaction” Chun-Hsien Lin, Ching-Lin Shyu, Zong-Yen Wu, Chao-Min Wang, Shiow-Her Chiou, Jiann-Yeu Chen, Shu-Ying Tseng, Ting-Er Lin, Yi-Po Yuan, Shu-Peng Ho, Kwong-Chung Tung, Frank Chiahung Mao, Han-Jung Lee and Wu-chun Tu investigate the antimicrobial activity and membrane disruption modes of the antimicrobial peptide mastoparan-AF against hemolytic Escherichia coli O157:H7.*

Based on the physicochemical properties, mastoparan-AF may potentially adopt a 3–11 amphipathic helix-type structure, with five to seven nonpolar or hydrophobic amino acid residues forming the hydrophobic face. E. coli O157:H7 and two diarrheagenic E. coli veterinary clinical isolates, which are highly resistant to multiple antibiotics, are sensitive to mastoparan-AF, with minimum inhibitory and bactericidal concentrations (MIC and MBC) ranging from 16 to 32 μg mL−1 for E. coli O157:H7 and four to eight μg mL−1 for the latter two isolates. *

Mastoparan-AF treatment, which correlates proportionally with membrane permeabilization of the bacteria, may lead to abnormal dents, large perforations or full opening at apical ends (hollow tubes), vesicle budding, and membrane corrugation and invagination forming irregular pits or pores on E. coli O157:H7 surface. In addition, mRNAs of prepromastoparan-AF and prepromastoparan-B share a 5′-poly(A) leader sequence at the 5′-UTR known for the advantage in cap-independent translation. *

This is the first report about the physicochemical adaptation of 3–11 amphipathic helices among mastoparans or antimicrobial peptides. *

Considering that E. coli O157:H7 and clinical isolates are highly resistant to multiple classes of antibiotics, mastoparan-AF, with little or mild effect on animal RBCs, could be an effective and alternative treatment to combat hemolytic E. coli O157:H7 and other pathogenic E. coli.*

The topography of bacteria was measured by a commercial atomic force microscope using NanoWorld Pointprobe® NCSTR AFM probes with a typical resonance frequency of 160 kHz and a typical spring constant of 7.4 N/m, respectively. For image quality, the scan rates of the tip were 0.3–0.6 Hz, with a resolution set of 512 by 256 pixels, and the feedback control parameters were optimized. *

Figure 5 from «Antimicrobial Peptide Mastoparan-AF Kills Multi-Antibiotic Resistant Escherichia coli O157:H7 via Multiple Membrane Disruption Patterns and Likely by Adopting 3–11 Amphipathic Helices to Favor Membrane Interaction» by Chun-Hsien Lin et al.:The topology of mastoparan-AF treated-hemolytic E. coli O157:H7 analyzed by AFM. (A) Two-dimensional (2D) and (B) three-dimensional (3D) images show smooth cell surfaces of untreated hemolytic E. coli O157:H7. (C) A 2D image of mastoparan-AF (32 μg mL−1)-treated hemolytic E. coli O157:H7. Abnormal perforations and dents on the surface of bacteria are indicated by arrows and arrowheads, respectively. The 3D images focusing on two highlighted areas of (C), respectively, reveal (D) a rough cell surface and (E) a hollow tube resulting from perforations at apical ends. (F) A 3D image shows a mastoparan-AF-treated bacterium with a budding vesicle. (G) A 3D image shows mastoparan-AF-treated bacteria with a wrinkled or rough surface. (H) Magnification of portion of (G) displays, in high resolution, the surface roughness of a mastoparan-AF-treated bacterium. The topography of bacteria was measured by a commercial atomic force microscope using NanoWorld Pointprobe® NCSTR AFM probes with a typical resonance frequency of 160 kHz and a typical spring constant of 7.4 N/m, respectively. For image quality, the scan rates of the tip were 0.3–0.6 Hz, with a resolution set of 512 by 256 pixels, and the feedback control parameters were optimized. * — Figure 5 from «Antimicrobial Peptide Mastoparan-AF Kills Multi-Antibiotic Resistant Escherichia coli O157:H7 via Multiple Membrane Disruption Patterns and Likely by Adopting 3–11 Amphipathic Helices to Favor Membrane Interaction» by Chun-Hsien Lin et al.:
The topology of mastoparan-AF treated-hemolytic E. coli O157:H7 analyzed by AFM. (A) Two-dimensional (2D) and (B) three-dimensional (3D) images show smooth cell surfaces of untreated hemolytic E. coli O157:H7. (C) A 2D image of mastoparan-AF (32 μg mL−1)-treated hemolytic E. coli O157:H7. Abnormal perforations and dents on the surface of bacteria are indicated by arrows and arrowheads, respectively. The 3D images focusing on two highlighted areas of (C), respectively, reveal (D) a rough cell surface and (E) a hollow tube resulting from perforations at apical ends. (F) A 3D image shows a mastoparan-AF-treated bacterium with a budding vesicle. (G) A 3D image shows mastoparan-AF-treated bacteria with a wrinkled or rough surface. (H) Magnification of portion of (G) displays, in high resolution, the surface roughness of a mastoparan-AF-treated bacterium.

*Chun-Hsien Lin, Ching-Lin Shyu, Zong-Yen Wu, Chao-Min Wang, Shiow-Her Chiou, Jiann-Yeu Chen, Shu-Ying Tseng, Ting-Er Lin, Yi-Po Yuan, Shu-Peng Ho, Kwong-Chung Tung, Frank Chiahung Mao, Han-Jung Lee and Wu-chun Tu
Antimicrobial Peptide Mastoparan-AF Kills Multi-Antibiotic Resistant Escherichia coli O157:H7 via Multiple Membrane Disruption Patterns and Likely by Adopting 3–11 Amphipathic Helices to Favor Membrane Interaction
Membranes 2023, 13(2), 251
DOI: https://doi.org/10.3390/membranes13020251

The article “Antimicrobial Peptide Mastoparan-AF Kills Multi-Antibiotic Resistant Escherichia coli O157:H7 via Multiple Membrane Disruption Patterns and Likely by Adopting 3–11 Amphipathic Helices to Favor Membrane Interaction” by Chun-Hsien Lin, Ching-Lin Shyu, Zong-Yen Wu, Chao-Min Wang, Shiow-Her Chiou, Jiann-Yeu Chen, Shu-Ying Tseng, Ting-Er Lin, Yi-Po Yuan, Shu-Peng Ho, Kwong-Chung Tung, Frank Chiahung Mao, Han-Jung Lee and Wu-chun Tu is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

Cross-sectional Kelvin probe force microscopy on III–V epitaxial multilayer stacks: challenges and perspectives

The development of photovoltaic (PV) technologies has progressed significantly over the past twenty years as a result of considerable advancements in solar cell device engineering and material science. *

As a consequence, solar cells have turned into complex structures containing numerous layers and interfaces. The capability to conduct local investigations at the nanoscale level that provide information on the electrical properties of materials and along physical interfaces is becoming crucial for solar photovoltaic device efficiency improvement. *

The capability to conduct local investigations at the nanoscale level that provide information on the electrical properties of materials and along physical interfaces is becoming crucial for solar photovoltaic device efficiency improvement. *

Multilayer III–V-based solar cells are complex devices consisting of many layers and interfaces. *

The study and the comprehension of the mechanisms that take place at the interfaces is crucial for efficiency improvement. *

Electrical measurements based on scanning probe microscopy (SPM) allow for the analysis of two-dimensional (2D) features at the surface and along a physical cross section of nanoscale semiconductor structures. *

Among the wide variety of SPM techniques available, Kelvin probe force microscopy (KPFM) is an application of the atomic force microscope (AFM) for the evaluation of the surface potential with nanometric resolution. KPFM is a valuable investigative approach for the study of work functions via the measurement of the contact potential difference VCPD, that is, the difference between the electrostatic potential at the surface of the investigated structure and that of the KPFM probe. *

In the article “Cross-sectional Kelvin probe force microscopy on III–V epitaxial multilayer stacks: challenges and perspectives” Mattia da Lisca, José Alvarez, James P. Connolly, Nicolas Vaissiere, Karim Mekhazni, Jean Decobert and Jean-Paul Kleider apply frequency-modulated Kelvin probe force microscopy (FM-KPFM) under ambient conditions to investigate the capability of this technique for the analysis of an InP/GaInAs(P) multilayer stack. *

KPFM reveals a strong dependence on the local doping concentration, allowing for the detection of the surface potential of layers with a resolution as low as 20 nm. *

The analysis of the surface potential allowed for the identification of space charge regions and, thus, the presence of several junctions along the stack. Furthermore, a contrast enhancement in the surface potential image was observed when KPFM was performed under illumination, which is analysed in terms of the reduction of surface band bending induced by surface defects by photogenerated carrier distributions. The analysis of the KPFM data was assisted by means of theoretical modelling simulating the energy bands profile and KPFM measurements. *

KPFM was performed using a scanning probe microscopy system under ambient conditions and operated in the frequency-modulated KPFM (FM-KPFM) mode using a two-pass scanning mode, where the second pass was performed at a constant distance of 10 nm from the sample surface. *

The FM-KPFM mode was chosen over the amplitude-modulation mode (AM-KPFM) since it is well known that it provides better spatial resolution. In particular, in AM-KPFM the electrical force between the tip and the sample is directly evaluated, whereas in FM-KPFM the gradient of the force is analysed. As a result, FM-KPFM is more sensitive to local tip apex–sample surface interactions; therefore, long-range electrostatic interactions of the cantilever are reduced, as well as the effect of parasitic capacitances. Additionally, in FM-KPFM, surface potential measurements are less dependent on the lift-height tip–sample distance than in AM-KPFM since this mode is less sensitive to static offsets induced by capacitive coupling or crosstalk. *

The laser beam deflection system in the author’s AFM employs a laser wavelength of 1310 nm, which is well below the bandgap of the sample; therefore, the parasitic laser absorption, which may interfere with the KPFM measurement, is reduced to negligible levels. Highly doped NanoWorld n+-Si ARROW-EFM tips (typical AFM tip radius < 25 nm) with a conductive Pt/Ir coating at a typical resonance frequency of 75 kHz were used. *

Figure 4 from “Cross-sectional Kelvin probe force microscopy on III–V epitaxial multilayer stacks: challenges and perspectives” by Mattia da Lisca et al :
KPFM measurement under ambient conditions on the surface cross section of the sample under illumination: (a) topography and (b) VCPD image. A vertical coloured bar is included to ease the identification of the different layers. The profile in (c) corresponds to the region identified by the dotted white segments in (b), each point of the profile (vertical) direction being an average of 207 points over a width of 0.7 μm along the x axis. Several regions along the structure have been highlighted using different colours (see text). The black arrow indicates the space charge region at the InP:nid/InP:Zn interface.

*Mattia da Lisca, José Alvarez, James P. Connolly, Nicolas Vaissiere, Karim Mekhazni, Jean Decobert and Jean-Paul Kleider
Cross-sectional Kelvin probe force microscopy on III–V epitaxial multilayer stacks: challenges and perspectives
Beilstein Journal of Nanotechnology 2023, 14, 725–737
DOI: https://doi.org/10.3762/bjnano.14.59

The article “Cross-sectional Kelvin probe force microscopy on III–V epitaxial multilayer stacks: challenges and perspectives” by Mattia da Lisca, José Alvarez, James P. Connolly, Nicolas Vaissiere, Karim Mekhazni, Jean Decobert and Jean-Paul Kleider is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response

Liquid–liquid phase-separated biomolecular condensates, liquid droplets play an important role in many biological processes, such as gene expression, protein translation, stress response, and protein degradation, by incorporating a variety of RNA and client proteins into their interior depending on the intracellular context. *

Autophagy is involved in the degradation of several cytoplasmic liquid droplets, including stress granules and P bodies, and defects in this process are thought to cause transition of these droplets to the solid phase, resulting in the development of intractable diseases such as neurodegenerative disorders and cancer. *

Of the droplets that have a unique biological function and are degraded by autophagy, p62 bodies (also called p62 droplets) are liquid droplets formed by liquid–liquid phase separation (LLPS) of p62 and its binding partners, ubiquitinated proteins. *

p62 bodies are involved in the regulation of intracellular proteostasis through their own autophagic degradation, and also contribute to the regulation of the major stress-response mechanism by sequestration of a client protein, kelch-like ECH-associated protein 1 (KEAP1). *

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid–liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. *

However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. *

In the article “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” Ryo Ikeda, Daisuke Noshiro, Hideaki Morishita, Shuhei Takada, Shun Kageyama, Yuko Fujioka, Tomoko Funakoshi, Satoko Komatsu-Hirota, Ritsuko Arai, Elena Ryzhii, Manabu Abe, Tomoaki Koga, Hozumi Motohashi, Mitsuyoshi Nakao, Kenji Sakimura, Arata Horii, Satoshi Waguri, Yoshinobu Ichimura, Nobuo N Noda and Masaaki Komatsu identify ULK1 as a kinase responsible for the phosphorylation of p62. *

ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. *

These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. *

The authors’ results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process. *

To clarify whether the ULK1 kinase itself has an effect on the physical properties and physiological role of p62 bodies, Ryo Ikeda et al. first studied the physical interaction of p62 with ULK1 or its yeast homolog Atg1 using high-speed atomic force microscopy (HS-AFM). *

HS-AFM of p62 (268–440 aa) visualized a homodimeric structure, mediated by the dimerization of the UBA domain, that formed a hammer-shaped structure with IDRs wrapped around each other. *

HS-AFM images were acquired in tapping mode using a sample-scanning HS-AFM instrument. NanoWorld Ultra-Short Cantilevers of the USC-F1.2-k0.15 AFM probe type were used. ( ~7 μm long, ~2 μm wide, and ~0.08 μm thick with electron beam-deposited (EBD) tips (tip radius < 10 nm). Their resonant frequency and spring constant were 1.2 MHz in air and 0.15 N/m, respectively.*

Figure EV1 from “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” by Ryo Ikeda et al.:HS-AFM observation of SNAP-ULK1 and p62 (268–440 aa), and complex of SNAP-Atg1/p62 (268–440 aa) A, B. Successive HS-AFM images of SNAP-ULK1 (A) and p62_268–440 (B). Height scale: 0–4.4 nm (A), 0–3.4 nm (B); scale bar: 20 nm (A, B). C. Successive HS-AFM images of p62_268–440 with SNAP-Atg1. Height scale: 0–3.6 nm; scale bar: 30 nm. D. Schematics showing the molecular characteristics determined by HS-AFM. Gray spheres, globular domains consisting of N-terminal KD and C-terminal MIT of Atg1; pink spheres, globular domains consisting of C-terminal UBA domain of p62; blue thick solid lines, IDRs. NanoWorld Ultra-Short Cantilevers of the USC-F1.2-k0.15 AFM probe type were used. — Figure EV1 from “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” by Ryo Ikeda et al.:
HS-AFM observation of SNAP-ULK1 and p62 (268–440 aa), and complex of SNAP-Atg1/p62 (268–440 aa)
A, B. Successive HS-AFM images of SNAP-ULK1 (A) and p62_268–440 (B). Height scale: 0–4.4 nm (A), 0–3.4 nm (B); scale bar: 20 nm (A, B).
C. Successive HS-AFM images of p62_268–440 with SNAP-Atg1. Height scale: 0–3.6 nm; scale bar: 30 nm.
D. Schematics showing the molecular characteristics determined by HS-AFM. Gray spheres, globular domains consisting of N-terminal KD and C-terminal MIT of Atg1; pink spheres, globular domains consisting of C-terminal UBA domain of p62; blue thick solid lines, IDRs.

*Ryo Ikeda, Daisuke Noshiro, Hideaki Morishita, Shuhei Takada, Shun Kageyama, Yuko Fujioka, Tomoko Funakoshi, Satoko Komatsu-Hirota, Ritsuko Arai, Elena Ryzhii, Manabu Abe, Tomoaki Koga, Hozumi Motohashi, Mitsuyoshi Nakao, Kenji Sakimura, Arata Horii, Satoshi Waguri, Yoshinobu Ichimura, Nobuo N Noda and Masaaki Komatsu
Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response
The EMBO Journal (2023)42:e113349
DOI: https://doi.org/10.15252/embj.2022113349

The article “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” by Ryo Ikeda, Daisuke Noshiro, Hideaki Morishita, Shuhei Takada, Shun Kageyama, Yuko Fujioka, Tomoko Funakoshi, Satoko Komatsu-Hirota, Ritsuko Arai, Elena Ryzhii, Manabu Abe, Tomoaki Koga, Hozumi Motohashi, Mitsuyoshi Nakao, Kenji Sakimura, Arata Horii, Satoshi Waguri, Yoshinobu Ichimura, Nobuo N Noda and Masaaki Komatsu is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.