Peptide-Lipid Huge Toroidal Pore, a New Antimicrobial Mechanism Mediated by a Lactococcal Bacteriocin, Lacticin Q

ABSTRACT Lacticin Q is a pore-forming bacteriocin produced by Lactococcus lactis QU 5, and its antimicrobial activity is in the nanomolar range. Lacticin Q induced calcein leakage from negatively charged liposomes. However, no morphological changes in the liposomes were observed by light scattering. Concomitantly with the calcein leakage, lacticin Q was found to translocate from the outer to the inner leaflet of the liposomes, after it initially bound to the membrane within 2 s. Lacticin Q also induced lipid flip-flop. These results reveal that the antimicrobial mechanism of lacticin Q can be described by the toroidal pore model. This is the first report of a bacteriocin of gram-positive bacteria that forms a toroidal pore. From liposomes, lacticin Q leaked fluorescence-labeled dextran with a diameter of 4.6 nm. In addition, lacticin Q caused the leakage of small proteins, such as the green fluorescent protein, from live bacterial cells. There are no other reports of antimicrobial peptides that exhibit protein leakage properties. The proposed pore formation model of lacticin Q is as follows: (i) quick binding to outer membrane leaflets; (ii) the formation of at least 4.6-nm pores, causing protein leakage with lipid flip-flop; and (iii) the migration of lacticin Q molecules from the outer to the inner membrane leaflets. Consequently, we termed the novel pore model in the antimicrobial mechanism of lacticin Q a “huge toroidal pore.”

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Classification of antimicrobial mechanism of action using bacterial time-lapse imaging

10.1101/2021.09.08.459470 ◽

2021 ◽

Author(s):

Xudong Ouyang ◽

Jelmer Hoeksma ◽

Tjalling K. Siersma ◽

Leendert W. Hamoen ◽

Jeroen den Hertog

Keyword(s):

Mechanism Of Action ◽

Morphological Changes ◽

Time Lapse ◽

Basic Knowledge ◽

Imaging Method ◽

Fungal Culture ◽

Bacterial Cells ◽

Simple Method ◽

Antimicrobial Mechanism

AbstractAntimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. Using this method, we found that the poorly studied antimicrobial, harzianic acid, a secondary metabolite that we purified from the fungal culture of Oidiodendron flavum, targets the cell envelope. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.

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Faculty Opinions recommendation of RNA visualization in live bacterial cells using fluorescent protein complementation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718145205.793485382 ◽

2013 ◽

Author(s):

Luc Jaeger

Keyword(s):

Fluorescent Protein ◽

Bacterial Cells ◽

Protein Complementation ◽

Live Bacterial Cells

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Zinc Oxide and Silver Nanoparticle Effects on Intestinal Bacteria

Materials ◽

10.3390/ma14102489 ◽

2021 ◽

Vol 14 (10) ◽

pp. 2489

Author(s):

Ami Yoo ◽

Mengshi Lin ◽

Azlin Mustapha

Keyword(s):

Electron Microscopy ◽

Zinc Oxide ◽

Morphological Changes ◽

Intestinal Bacteria ◽

Bacterial Cells ◽

Ag Nps ◽

Zno Nps ◽

Bifidobacterium Animalis ◽

Transmission Electron

The application of nanoparticles (NPs) for food safety is increasingly being explored. Zinc oxide (ZnO) and silver (Ag) NPs are inorganic chemicals with antimicrobial and bioactive characteristics and have been widely used in the food industry. However, not much is known about the behavior of these NPs upon ingestion and whether they inhibit natural gut microflora. The objective of this study was to investigate the effects of ZnO and Ag NPs on the intestinal bacteria, namely Escherichia coli, Lactobacillus acidophilus, and Bifidobacterium animalis. Cells were inoculated into tryptic soy broth or Lactobacilli MRS broth containing 1% of NP-free solution, 0, 12, 16, 20 mM of ZnO NPs or 0, 1.8, 2.7, 4.6 mM Ag NPs, and incubated at 37 °C for 24 h. The presence and characterization of the NPs on bacterial cells were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDS). Membrane leakage and cell viability were assessed using a UV-visible spectrophotometer and confocal electron microscope, respectively. Numbers of treated cells were within 1 log CFU/mL less than those of the controls for up to 12 h of incubation. Cellular morphological changes were observed, but many cells remained in normal shapes. Only a small amount of internal cellular contents was leaked due to the NP treatments, and more live than dead cells were observed after exposure to the NPs. Based on these results, we conclude that ZnO and Ag NPs have mild inhibitory effects on intestinal bacteria.

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Regulation of Mineralocorticoid Receptor Expression during Neuronal Differentiation of Murine Embryonic Stem Cells

Endocrinology ◽

10.1210/en.2009-0753 ◽

2010 ◽

Vol 151 (5) ◽

pp. 2244-2254 ◽

Cited By ~ 19

Author(s):

Mathilde Munier ◽

Geri Meduri ◽

Say Viengchareun ◽

Philippe Leclerc ◽

Damien Le Menuet ◽

...

Keyword(s):

Neuronal Differentiation ◽

Mineralocorticoid Receptor ◽

Fluorescent Protein ◽

Morphological Changes ◽

Critical Role ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Receptor Expression ◽

Neuronal Markers ◽

Mature Neurons

Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem cell (ES) lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene green fluorescent protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted us to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites), and the increase in the expression of neuronal markers (nestin, β-tubulin III, and microtubule-associated protein-2) as demonstrated by immunocytochemistry and quantitative PCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively, and correlated with MR expression. Finally, although progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by small interfering RNA. In conclusion, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases.

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Cdk5 mediates changes in morphology and promotes apoptosis of astrocytoma cells in response to heat shock

Journal of Cell Science ◽

10.1242/jcs.114.6.1145 ◽

2001 ◽

Vol 114 (6) ◽

pp. 1145-1153 ◽

Cited By ~ 1

Author(s):

C. Gao ◽

S. Negash ◽

H.S. Wang ◽

D. Ledee ◽

H. Guo ◽

...

Keyword(s):

Heat Shock ◽

Cell Line ◽

Fluorescent Protein ◽

Morphological Changes ◽

Normal Growth ◽

Dominant Negative ◽

Specific Expression ◽

Cell Matrix ◽

Dominant Negative Mutation ◽

U373 Cells

The cyclin-dependent kinase member, Cdk5, is expressed in a variety of cell types, but neuron-specific expression of its activator, p35, is thought to limit its activity to neurons. Here we demonstrate that both Cdk5 and p35 are expressed in the human astrocytoma cell line, U373. Cdk5 and p35 are present in the detergent-insoluble cytoskeletal fraction of this cell line and Cdk5 localizes to filopodia and vinculin-rich regions of cell-matrix contact in lamellopodia. When exposed to a 46(o)C heat shock, U373 cells change shape, lose cell-matrix contacts and show increased levels of apoptosis. To test whether Cdk5 activation might play a role in these events, U373 cells were stably transfected with histidine-tagged or green fluorescent protein-tagged constructs of Cdk5 or a dominant negative mutation, Cdk5T33. Under normal growth conditions, growth characteristics of the stably transfected lines were indistinguishable from untransfected U373 cells and Cdk5 localization was not changed. However, when subjected to heat shock, cells stably transfected with Cdk5-T33 remained flattened, showed little loss of cell-matrix adhesion, and exhibited significantly lower levels of apoptosis. In contrast, cells that overexpressed wild-type Cdk5 showed morphological changes similar to those seen in untransfected U373 cells in response to heat shock and had significantly higher levels of apoptosis. Heat-shocked cells showed changes in p35 mobility and stability of the Cdk5/p35 complex consistent with endogenous Cdk5 activity. Together these findings suggest that endogenous Cdk5 activity may play a key role in regulating morphology, attachment, and apoptosis in U373 cells, and raise the possibility that Cdk5 may be a general regulator of cytoskeletal organization and cell adhesion in both neuronal and non-neuronal cells.

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Morphological Alterations of CAD Cells Overexpressing AKT1

Microscopy and Microanalysis ◽

10.1017/s1431927620017821 ◽

2020 ◽

Vol 26 (S2) ◽

pp. 1354-1358

Author(s):

James Wachira

Keyword(s):

Signal Transduction ◽

Plasma Membrane ◽

Cell Survival ◽

Fluorescent Protein ◽

Morphological Changes ◽

Protein Localization ◽

Signal Transduction Pathways ◽

Morphological Alterations ◽

Cellular Models ◽

Cad Cells

AbstractCAD cells are neuronal cells used in studies of cell differentiation and in cellular models of neuropathology. When cultured in differentiation medium, CAD cells exhibit characteristics of mature neurons including the generation of action potential. In addition to being a central signaling kinase in cell survival, AKT1 plays important roles in the nervous system including neuroplasticity and this study examined the localization of exogenous AKT1 in CAD cells. Neuropeptides modulate many signal transduction pathways and melacortins are implicated in regulating growth factor signal transduction pathways, including the PI3K/AKT pathway. AKT1-DsReD was transfected into CAD cells that were stably expressing melanocortin 3-receptor-GFP (MC3R-GFP), a G-protein coupled receptor. The cells were imaged with confocal microscopy to determine the fluorescent protein localization patterns. AKT1-DsRed was predominantly localized in the cytoplasm and the nucleus. Further, expression of exogenous AKT1 in these cell lines led to morphological changes reminiscent of apoptosis. As expected, MC3R-GFP localized to the plasma membrane but it internalized upon cell stimulation with the cognate ligand. In limited areas of the plasma membrane, AKT1-DsRed and MC3R-GFP were colocalized. In conclusion, quantitative studies to understand the role of relative levels of AKT1 in determining cell survival are needed.

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The effect of exogenous RNA extract on chick embryo fibroblasts in vitro

Development ◽

10.1242/dev.23.2.509 ◽

1970 ◽

Vol 23 (2) ◽

pp. 509-517

Author(s):

A. Sann ◽

D. Sharp ◽

J. McKenzie

Keyword(s):

Cell Differentiation ◽

Chick Embryo ◽

Morphological Changes ◽

Bacterial Cells ◽

Baby Hamster Kidney ◽

Exogenous Rna ◽

Embryo Fibroblasts ◽

Stimulation Of ◽

Rna Extract

It is extremely difficult, if not impossible, to reconcile the conflicting claims of those who have treated different cells and tissues with exogenous RNA. Some authors (e.g. Niu, Cordova & Niu, 1961; Niu, Cordova & Radbill, 1962) maintain that RNA extracts alter the course of cell differentiation to conform in morphological terms to the source of the RNA; in the same vein, Amos, Askonas & Soeiro (1964) have shown that, under certain conditions, RNA from mouse and bacterial cells can stimulate chick embryo fibroblasts to synthesize protein related antigenically to the origin of the RNA. Shepley, Ambrose & Kirby (1965), however, obtained stimulation of growth with permanent morphological changes in baby hamster kidney fibroblasts by the addition of RNA from a variety of sources.

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Membrane Remodeling by the Lytic Fragment of SticholysinII: Implications for the Toroidal Pore Model

Biophysical Journal ◽

10.1016/j.bpj.2019.09.018 ◽

2019 ◽

Vol 117 (9) ◽

pp. 1563-1576 ◽

Cited By ~ 3

Author(s):

Haydee Mesa-Galloso ◽

Pedro A. Valiente ◽

Mario E. Valdés-Tresanco ◽

Raquel F. Epand ◽

Maria E. Lanio ◽

...

Keyword(s):

Membrane Remodeling ◽

Pore Model ◽

Toroidal Pore

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Induced Expression of the Heat Shock Protein Genes uspA and grpE during Starvation at Low Temperatures and Their Influence on Thermal Resistance of Escherichia coli O157:H7

Journal of Food Protection ◽

10.4315/0362-028x-66.11.2045 ◽

2003 ◽

Vol 66 (11) ◽

pp. 2045-2050 ◽

Cited By ~ 25

Author(s):

YI ZHANG ◽

MANSEL W. GRIFFITHS

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

Stress Responses ◽

Thermal Tolerance ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Bacterial Cells ◽

Induced Expression ◽

Green Fluorescent ◽

Shock Proteins

Heat shock proteins play an important role in protecting bacterial cells against several stresses, including starvation. In this study, the promoters for two genes encoding heat shock proteins involved in many stress responses, UspA and GrpE, were fused with the green fluorescent protein (gfp) gene. Thus, the expression of the two genes could be quantified by measuring the fluorescence emitted by the cells under different environmental conditions. The heat resistance levels of starved and nonstarved cells during storage at 5, 10, and 37°C were compared with the levels of expression of the uspA and grpE genes. D52-values (times required for decimal reductions in count at 52°C) increased by 11.5, 14.6, and 18.5 min when cells were starved for 3 h at 37°C, for 24 h at 10°C, and for 2 days at 5°C, respectively. In all cases, these increases were significant (P < 0.01), indicating that the stress imposed by starvation altered the ability of E. coli O157:H7 to survive subsequent heat treatments. Thermal tolerance was correlative with the induction of UspA and GrpE. At 5°C, the change in the thermal tolerance of the pathogen was positively linked to the induced expression of the grpE gene but negatively related to the expression of the uspA gene. The results obtained in this study indicate that UspA plays an important role in starvation-induced thermal tolerance at 37°C but that GrpE may be more involved in regulating this response at lower temperatures. An improvement in our understanding of the molecular mechanisms involved in these cross-protection responses may make it possible to devise strategies to limit their effects.

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