scholarly journals Prediction and Activity of a Cationic α-Helix Antimicrobial Peptide ZM-804 from Maize

2021 ◽  
Vol 22 (5) ◽  
pp. 2643
Author(s):  
Mohamed F. Hassan ◽  
Abdelrahman M. Qutb ◽  
Wubei Dong
Keyword(s):  
Cdna Library ◽  
Pseudomonas Syringae ◽  
Inbred Line ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Pathogenic Bacteria ◽  
Antimicrobial Activities ◽  
Confocal Laser ◽  
In Planta ◽  
Minimal Inhibitory Concentrations

Antimicrobial peptides (AMPs) are small molecules consisting of less than fifty residues of amino acids. Plant AMPs establish the first barrier of defense in the innate immune system in response to invading pathogens. The purpose of this study was to isolate new AMPs from the Zea mays L. inbred line B73 and investigate their antimicrobial activities and mechanisms against certain essential plant pathogenic bacteria. In silico, the Collection of Anti-Microbial Peptides (CAMPR3), a computational AMP prediction server, was used to screen a cDNA library for AMPs. A ZM-804 peptide, isolated from the Z. mays L. inbred line B73 cDNA library, was predicted as a new cationic AMP with high prediction values. ZM-804 was tested against eleven pathogens of Gram-negative and Gram-positive bacteria and exhibited high antimicrobial activities as determined by the minimal inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). A confocal laser scanning microscope observation showed that the ZM-804 AMP targets bacterial cell membranes. SEM and TEM images revealed the disruption and damage of the cell membrane morphology of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato (Pst) DC3000 caused by ZM-804. In planta, ZM-804 demonstrated antimicrobial activity and prevented the infection of tomato plants by Pst DC3000. Moreover, four virulent phytopathogenic bacteria were prevented from inducing hypersensitive response (HR) in tobacco leaves in response to low ZM-804 concentrations. ZM-804 exhibits low hemolytic activity against mouse red blood cells (RBCs) and is relatively safe for mammalian cells. In conclusion, the ZM-804 peptide has a strong antibacterial activity and provides an alternative tool for plant disease control. Additionally, the ZM-804 peptide is considered a promising candidate for human and animal drug development.

scholarly journals Impact of Temperature on In Planta Expression of Genes Involved in Synthesis of the Pseudomonas syringae Phytotoxin Coronatine

2004 ◽  
Vol 17 (10) ◽  
pp. 1095-1102 ◽  
Author(s):  
Helge Weingart ◽  
Stephan Stubner ◽  
Alexander Schenk ◽  
Matthias S. Ullrich
Keyword(s):  
Pseudomonas Syringae ◽  
Laser Scanning ◽  
Minimal Medium ◽  
Laser Scanning Microscopy ◽  
Model Organisms ◽  
Confocal Laser ◽  
Dependent Manner ◽  
In Planta ◽  
Temperature Dependent

Coronatine (COR) is a chlorosis-inducing phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Confocal laser scanning microscopy was used to investigate in vitro and in planta expression of COR genes by two model organisms, P. syringae pv. glycinea PG4180, a pathogen of soybean, and P. syringae pv. tomato DC3000, a pathogen of tomato and crucifers. Previously, it was shown in vitro that the cma operon involved in COR synthesis in PG4180 is expressed in a temperature-dependent manner, with maximal rates at 18°C and low activity at 28°C. However, nothing was known about the influence of temperature on the expression of COR biosynthetic genes in planta. Therefore, transcriptional fusions of the PG4180 and DC3000 cma promoter regions to a promoterless egfp gene were constructed and expressed in both P. syringae strains. The fluorescence patterns in response to temperature during growth of a strain in vitro were consistent with its COR production and the cma transcript abundance as revealed by RNA dot blot hybridization. Quantification of fluorescence indicated that cma promoter activity was dependent on the genetic background of the host strain. Expression of cma∷egfp in PG4180 was temperature-dependent in minimal medium as well as inside the plant tissue. In contrast, transcription of the cma operon was not significantly affected by temperature in DC3000. However, cells of DC3000 harboring the cma∷egfp fusions showed higher levels of fluorescence when recovered from infected host plants compared with cells grown in minimal medium. These results indicate that the signals for induction of COR biosynthesis differ significantly in PG4180 and DC3000.

scholarly journals A Gene in the Pseudomonas syringae pv. tomato Hrp Pathogenicity Island Conserved Effector Locus, hopPtoA1, Contributes to Efficient Formation of Bacterial Colonies in Planta and Is Duplicated Elsewhere in the Genome

2002 ◽  
Vol 15 (10) ◽  
pp. 1014-1024 ◽  
Author(s):  
J. L. Badel ◽  
A. O. Charkowski ◽  
W.-L. Deng ◽  
A. Collmer
Keyword(s):  
Pseudomonas Syringae ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
In Planta ◽  
Tomato Dc3000 ◽  
Green Fluorescent

The ability of Pseudomonas syringae to grow in planta is thought to be dependent upon the Hrp (type III secretion) system and multiple effector proteins that this system injects into plant cells. ORF5 in the conserved effector locus of the P. syringae pv. tomato DC3000 Hrp pathogenicity island was shown to encode a Hrp-secreted protein and to have a similarly secreted homolog encoded in an effector-rich pathogenicity island located elsewhere in the genome. These putative effector genes were designated hopPtoA1 and hopPtoA2, respectively. DNA gel blot analysis revealed that sequences hybridizing with hopPtoA1 were widespread among P. syringae pathovars, and some strains, like DC3000, appear to have two copies of the gene. uidA transcriptional fusions revealed that expression of hopPtoA1 and hopPtoA2 can be activated by the HrpL alternative sigma factor. hopPtoA1 and hopPtoA1/hopPtoA2 double mutants were not obviously different from wild-type P. syringae pv. tomato DC3000 in their ability to produce symptoms or to increase their total population size in host tomato and Arabidopsis leaves. However, confocal laser-scanning microscopy of GFP (green fluorescent protein)-labeled bacteria in Arabidopsis leaves 2 days after inoculation revealed that the frequency of undeveloped individual colonies was higher in the hopPtoA1 mutant and even higher in the hopPtoA1/hopPtoA2 double mutant. These results suggest that hopPtoA1 and hopPtoA2 contribute redundantly to the formation of P. syringae pv. tomato DC3000 colonies in Arabidopsis leaves.

scholarly journals Super-antibiofilm effect of N2 plasma treated buffer (NPB) against plant pathogenic bacterium

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Hyemi Seo ◽  
Jisoo Hong ◽  
Taeyeol Kim ◽  
Won ll Choi ◽  
Daekyung Sung ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Laser Scanning ◽  
Pathogenic Bacteria ◽  
Pathogenic Bacterium ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Antibiofilm Activity ◽  
Plant Pathogenic Bacterium ◽  
Bacterial Inactivation ◽  
Imaging Data

AbstractControlling of biofilms formation in numerous pathogenic bacteria is one of the most difficult tasks in the control of bacterial diseases. Plasma has attracted extensive attention due to their potential applications for effective inhibiting of biofilm. Recently, plasma-activated water (PAW) has developed as an alternative method for bacterial inactivation and disinfection of foods owing to advantages of more convenient and efficient storage and transportation than direct plasma application. However, most previous studies about PAW have only focused on the improvement of its antibacterial effect instead of antibiofilm activity. Therefore, we report the development of N2 plasma treated buffer (NPB) and the super-antibiofilm effect of NPB against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) as a plant pathogenic bacterium. Scavenger assays using various antioxidants revealed that reactive oxygen species were involved in the inhibitory cellular actions of NPB, with H2O2 and singlet oxygen proving essential for bacterial death. Intensive analysis of NPB, stored at different periods and temperatures, showed that the antimicrobial efficacy was well maintained for 3 months at − 80 °C. Importantly, further studies showed that NPB effectively inhibited not only the growth of planktonic Pst DC3000 but also biofilm formation. The remarkable inhibition on the biofilm was analyzed and visualized using LIVE/DEAD viability assays and confocal laser scanning microscopy (CLSM) imaging. The 3D CLSM imaging data revealed that the bactericidal activity of NPB was permeable enough to affect the cells embedded inside the biofilm. This prominent permeability could be a crucial feature of NPB contributing to effective super-antibiofilm.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

scholarly journals One-step preparation of antimicrobial silicone materials based on PDMS and salicylic acid: insights from spatially and temporally resolved techniques

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Luca Barbieri ◽  
Ioritz Sorzabal Bellido ◽  
Alison J. Beckett ◽  
Ian A. Prior ◽  
Jo Fothergill ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Laser Scanning ◽  
Pathogenic Bacteria ◽  
Pharmaceutical Products ◽  
Laser Scanning Microscopy ◽  
Wound Dressings ◽  
Confocal Laser ◽  
Vis Spectroscopy ◽  
Wide Range ◽  
One Step

AbstractIn this work, we introduce a one-step strategy that is suitable for continuous flow manufacturing of antimicrobial PDMS materials. The process is based on the intrinsic capacity of PDMS to react to certain organic solvents, which enables the incorporation of antimicrobial actives such as salicylic acid (SA), which has been approved for use in humans within pharmaceutical products. By combining different spectroscopic and imaging techniques, we show that the surface properties of PDMS remain unaffected while high doses of the SA are loaded inside the PDMS matrix. The SA can be subsequently released under physiological conditions, delivering a strong antibacterial activity. Furthermore, encapsulation of SA inside the PDMS matrix ensured a diffusion-controlled release that was tracked by spatially resolved Raman spectroscopy, Attenuated Total Reflectance IR (ATR-IR), and UV-Vis spectroscopy. The biological activity of the new material was evaluated directly at the surface and in the planktonic state against model pathogenic bacteria, combining confocal laser scanning microscopy, electron microscopy, and cell viability assays. The results showed complete planktonic inhibition for clinically relevant strains of Staphylococcus aureus and Escherichia coli, and a reduction of up to 4 orders of magnitude for viable sessile cells, demonstrating the efficacy of these surfaces in preventing the initial stages of biofilm formation. Our approach adds a new option to existing strategies for the antimicrobial functionalisation of a wide range of products such as catheters, wound dressings and in-dwelling medical devices based on PDMS.

scholarly journals Biosensors Used for Epifluorescence and Confocal Laser Scanning Microscopies to Study Dickeya and Pectobacterium Virulence and Biocontrol

2021 ◽  
Vol 9 (2) ◽  
pp. 295
Author(s):  
Yvann Bourigault ◽  
Andrea Chane ◽  
Corinne Barbey ◽  
Sylwia Jafra ◽  
Robert Czajkowski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Infected Plant ◽  
Soft Rot ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Cell Physiology ◽  
In Planta ◽  
Reporter System ◽  
Cell Wall Lysis

Promoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future.

scholarly journals PGC-1-Related Coactivator, a Novel, Serum-Inducible Coactivator of Nuclear Respiratory Factor 1-Dependent Transcription in Mammalian Cells

2001 ◽  
Vol 21 (11) ◽  
pp. 3738-3749 ◽  
Author(s):  
Ulf Andersson ◽  
Richard C. Scarpulla
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Peroxisome Proliferator ◽  
Nuclear Respiratory Factor ◽  
Nuclear Respiratory Factor 1 ◽  
Cell Extracts

ABSTRACT The thermogenic peroxisome proliferator-activated receptor γ (PPAR-γ) coactivator 1 (PGC-1) has previously been shown to activate mitochondrial biogenesis in part through a direct interaction with nuclear respiratory factor 1 (NRF-1). In order to identify related coactivators that act through NRF-1, we searched the databases for sequences with similarities to PGC-1. Here, we describe the first characterization of a 177-kDa transcriptional coactivator, designated PGC-1-related coactivator (PRC). PRC is ubiquitously expressed in murine and human tissues and cell lines; but unlike PGC-1, PRC was not dramatically up-regulated during thermogenesis in brown fat. However, its expression was down-regulated in quiescent BALB/3T3 cells and was rapidly induced by reintroduction of serum, conditions where PGC-1 was not detected. PRC activated NRF-1-dependent promoters in a manner similar to that observed for PGC-1. Moreover, NRF-1 was immunoprecipitated from cell extracts by antibodies directed against PRC, and both proteins were colocalized to the nucleoplasm by confocal laser scanning microscopy. PRC interacts in vitro with the NRF-1 DNA binding domain through two distinct recognition motifs that are separated by an unstructured proline-rich region. PRC also contains a potent transcriptional activation domain in its amino terminus adjacent to an LXXLL motif. The spatial arrangement of these functional domains coincides with those found in PGC-1, supporting the conclusion that PRC and PGC-1 are structurally and functionally related. We conclude that PRC is a functional relative of PGC-1 that operates through NRF-1 and possibly other activators in response to proliferative signals.

Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs

1996 ◽  
Vol 109 (2) ◽  
pp. 367-377 ◽  
Author(s):  
H. Dutartre ◽  
J. Davoust ◽  
J.P. Gorvel ◽  
P. Chavrier
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Rho Gtpase ◽  
Laser Scanning Microscopy ◽  
Regulatory Subunit ◽  
Confocal Laser ◽  
Complex Architectures ◽  
Immunofluorescence Labelling

In mammalian cells, Rho GTPases control the reorganisation of the actin cytoskeleton in response to growth factors. In the cytoplasm, the polymerisation of actin filaments and their organisation into complex architectures is orchestrated by numerous proteins which act either directly, by interacting with actin, or by producing secondary messengers which serve as mediators between signal transduction pathways and the microfilament organisation. We sought to determine whether the intracellular distribution of some of these regulatory components may be controlled by the Rho GTPase CDC42Hs. With this aim, we have established HeLa-derived human cell lines in which expression of a constitutively activated mutant of CDC42Hs is inducible. Morphological analysis by immunofluorescence labelling and confocal laser scanning microscopy revealed a massive reorganisation of F-actin in cortical microspikes as well as podosome-like structures located at the ventral face of the cells. Concomitantly, the cells became giant and multinucleate indicating that cytokinesis was impaired. The actin bundling protein T-plastin, the vasodilatator-stimulated phosphoprotein (VASP), a profilin ligand, as well as the 85 kDa regulatory subunit of the phosphoinosite 3-kinase redistributed with F-actin into the CDC42Hs-induced structures.

scholarly journals Bactericidal compounds controlling growth of the plant pathogen pseudomonas syringae pv. actinidiae, which forms biofilms composed of a novel exopolysaccharide

2020 ◽  
Author(s):  
S Ghods ◽  
Ian Sims ◽  
MF Moradali ◽  
BHA Rehma
Keyword(s):  
Pseudomonas Syringae ◽  
Chlorine Dioxide ◽  
Laser Scanning ◽  
Extracellular Polymeric Substances ◽  
Virulent Strain ◽  
Bactericidal Effect ◽  
Cell System ◽  
Laser Scanning Microscopy ◽  
Base Layer ◽  
Confocal Laser

© 2015, American Society for Microbiology. Pseudomonas syringae pv. actinidiae is the major cause of bacterial canker and is a severe threat to kiwifruit production worldwide. Many aspects of the disease caused by P. syringae pv. actinidiae, such as the pathogenicity-relevant formation of a biofilm composed of extracellular polymeric substances (EPSs), are still unknown. Here, a highly virulent strain of P. syringae pv. actinidiae, NZ V-13, was studied with respect to biofilm formation and architecture using a flow cell system combined with confocal laser scanning microscopy. The biofilm formed by P. syringae pv. actinidiae NZ V-13 was heterogeneous, consisting of a thin cellular base layer 5 μm thick and microcolonies with irregular structures. The major component of the EPSs produced by P. syringae pv. actinidiae NZ V-13 bacteria was isolated and identified to be an exopolysaccharide. Extensive compositional and structural analysis showed that rhamnose, fucose, and glucose were the major constituents, present at a ratio of 5:1.5:2. Experimental evidence that P. syringae pv. actinidiae NZ V-13 produces two polysaccharides, a branched α-D-rhamnan with side chains of terminal α-D-Fucf and an α-D-1,4-linked glucan, was obtained. The susceptibility of the cells in biofilms to kasugamycin and chlorine dioxide was assessed. About 64 and 73% of P. syringae pv. actinidiae NZ V-13 cells in biofilms were killed when kasugamycin and chlorine dioxide were used at 5 and 10 ppm, respectively. Kasugamycin inhibited the attachment of P. syringae pv. actinidiae NZ V-13 to solid surfaces at concentrations of 80 and 100 ppm. Kasugamycin was bacteriostatic against P. syringae pv. actinidiae NZ V-13 growth in the planktonic mode, with the MIC being 40 to 60 ppm and a bactericidal effect being found at 100 ppm. Here we studied the formation, architecture, and composition of P. syringae pv. actinidiae biofilms as well as used the biofilm as a model to assess the efficacies of bactericidal compounds.

Quantification of microtubule dynamics in living plant cells using fluorescence redistribution after photobleaching

1994 ◽  
Vol 107 (4) ◽  
pp. 775-784 ◽  
Author(s):  
J.M. Hush ◽  
P. Wadsworth ◽  
D.A. Callaham ◽  
P.K. Hepler
Keyword(s):  
Mammalian Cells ◽  
Laser Scanning ◽  
Dynamic Instability ◽  
Animal Cell ◽  
Plant Cells ◽  
Preprophase Band ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Turnover Rates ◽  
Living Plant

Microtubule (MT) turnover within the four principal MT arrays, the cortical array, the preprophase band, the mitotic spindle and the phragmoplast, has been measured in living stamen hair cells of Tradescantia that have been injected with fluorescent neurotubulin. Using the combined techniques of confocal laser scanning microscopy and fluorescence redistribution after photobleaching (FRAP), we report that the half-time of turnover in spindle MTs is t 1/2 = 31 +/- 6 seconds, which is in excellent agreement with previous measurements of turnover in animal cell spindles. Tradescantia interphase MTs, however, exhibit turnover rates (t 1/2 = 67 +/- seconds) that are some 3.4-fold faster than those measured in interphase mammalian cells, and thus are revealed as being highly dynamic. Preprophase band and phragmoplast MTs have turnover rates similar to those of interphase MTs in Tradescantia. The spatial and temporal aspects of the fluorescence redistribution after photobleaching in all four MT arrays are more consistent with subunit exchange by the mechanism of dynamic instability than treadmilling. This is the first quantification of MT dynamics in plant cells.

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