scholarly journals Membrane Vesicles of Pectobacterium as an Effective Protein Secretion System

2021 ◽  
Vol 22 (22) ◽  
pp. 12574
Author(s):  
Joanna Jonca ◽  
Malgorzata Waleron ◽  
Paulina Czaplewska ◽  
Aleksandra Bogucka ◽  
Aleksandra Steć ◽  
...  
Keyword(s):  
Ornamental Plants ◽  
Green Fluorescence Protein ◽  
Membrane Vesicles ◽  
Soft Rot ◽  
Secretion System ◽  
Medium Composition ◽  
Pathogenicity Test ◽  
Cargo Delivery ◽  
Periplasmic Proteins ◽  
Niche Competition

Bacteria of genus Pectobacterium are Gram-negative rods of the family Pectobacteriaceae. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are believed to play an important role in the pathogenicity and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium. The results indicate that the morphology and MVs production depend on growth medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produces large MVs (100–300 nm) and small vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in the MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to degrade pectates. What is more, the pathogenicity test indicated that the MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that the MVs of β-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that the MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that the MVs production in Pectobacterium strains, which overexpress a green fluorescence protein (GFP), is higher than in wild-type strains. Moreover, proteomic analysis revealed that the GFP was present in the MVs. Therefore, it is possible that protein sequestration into MVs might not be strictly limited to periplasmic proteins. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an effective secretion system.

scholarly journals Effects of Culture Conditions on the Antibacterial Activity of Streptomyces Spp. against Erwinia Spp. Causing Soft Rot Disease on Asparagus Officinalis

2021 ◽  
Vol 4 (2) ◽  
pp. 1077-1086
Author(s):  
Nguyen Thanh Trung ◽  
Nguyen Thi Van Anh ◽  
Tran Thi Dao ◽  
Nguyen Thanh Huyen ◽  
Pham Le Anh Minh ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Ornamental Plants ◽  
Soft Rot ◽  
Antibacterial Activities ◽  
Culture Conditions ◽  
Asparagus Officinalis ◽  
Effective Control ◽  
Soft Rot Disease ◽  
Streptomyces Spp ◽  
Rot Disease

Erwinia is a genus of Enterobacteriacea containing mostly pathogens, which cause soft rot disease in many ornamental plants and crops, including Asparagus officinalis. Chemical treatments to control Erwinia have lost their attractiveness because of the development of resistant strains and the negative impacts on the environment and human health. Therefore, the study of biological controls of soft rot disease has gained great importance. There are several types of microorganisms that show activity against Erwinia spp. such as Pseudomonas fluorescence, Bacillus subtilis, and Streptomyces spp. Among them, Streptomyces spp. are found to be the most effective control agents. In this study, 64 isolates of Streptomyces were screened for their antibacterial activity against Erwinia spp. The results indicated that 18 isolates showed an antagonistic reaction against Erwinia spp. Among them, isolate D5.1 showed the highest inhibition activity. In addition, the morphological and antibacterial activities of isolate D5.1 grown in different conditions were also characterized. 

scholarly journals Intracellular Delivery of DNA and Protein by a Novel Cell-Permeable Peptide Derived from DOT1L

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 217 ◽  
Author(s):  
Jingping Geng ◽  
Xiangli Guo ◽  
Lidan Wang ◽  
Richard Q. Nguyen ◽  
Fengqin Wang ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Green Fluorescence Protein ◽  
Cell Penetrating Peptides ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cellular Targets ◽  
Intracellular Release ◽  
Cargo Delivery ◽  
Future Drug ◽  
Cell Permeable Peptide

Cellular uptake and intracellular release efficiency of biomacromolecules is low because of hurdles in the cell membrane that result in limited access to intra-cellular targets with few functional effects. Cell-penetrating peptides (CPPs) act as cargo delivery vehicles to promote therapeutic molecule translocation. Here, we describe the novel CPP-Dot1l that not only penetrates by itself, but also mediates cargo translocation in cultured cells, as confirmed by fluorescence microscopy and fluorescence spectrophotometry. We conducted cytotoxicity assays and safety evaluations, and determined peptide-membrane interactions to understand the possible pathway for cargo translocation. Additional nucleic acid and covalently conjugated green fluorescence protein (GFP) studies mediated by CPP-Dot1l were conducted to show functional delivery potential. Results indicate that CPP-Dot1l is a novel and effective CPP due to its good penetrating properties in different cell lines and its ability to enter cells in a concentration-dependent manner. Its penetration efficiency can be prompted by DMSO pretreatment. In addition, not only can it mediate plasmid delivery, but CPP-Dot1l can also deliver GFP protein into cytosol. In conclusion, the findings of this study showed CPP-Dot1l is an attractive pharmaceutical and biochemical tool for future drug, regenerative medicine, cell therapy, gene therapy, and gene editing-based therapy development.

scholarly journals The outer membrane vesicles: Secretion system type zero

Traffic ◽  
2017 ◽  
Vol 18 (7) ◽  
pp. 425-432 ◽  
Author(s):  
Andrea Guerrero-Mandujano ◽  
Cecilia Hernández-Cortez ◽  
Jose Antonio Ibarra ◽  
Graciela Castro-Escarpulli
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Secretion System ◽  
Outer Membrane Vesicles ◽  
System Type

scholarly journals Soft Rot Disease Severity Is Affected by Potato Physiology and Pectobacterium taxa

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 232-241 ◽  
Author(s):  
Maria del Pilar Marquez-Villavicencio ◽  
Russell L. Groves ◽  
Amy O. Charkowski
Keyword(s):  
Ornamental Plants ◽  
Soft Rot ◽  
The United States ◽  
High Variability ◽  
Physiological Factors ◽  
Carotovorum Subsp ◽  
Rot Disease ◽  
Bacterial Growth Inhibition ◽  
Field Location

Pectobacterium species cause disease worldwide in many crop and ornamental plants, including potato. A new Pectobacterium subspecies, P. carotovorum subsp. brasiliensis was recently described in Brazil and later found in the United States, Israel, and South Africa. Its virulence traits and host range remain unknown. A comparison of three taxa commonly found on potato showed that both P. carotovorum subsp. carotovorum and subsp. brasiliensis are more aggressive in causing tuber and stem soft rot than P. atrosepticum. Also, despite bacterial growth inhibition in vitro of P. carotovorum subsp. carotovorum and P. atrosepticum strains by P. carotovorum subsp. brasiliensis, this new subspecies and P. carotovorum subsp. carotovorum are able to co-colonize in the same infected tissue. Both subspecies were motile in lesions. Pathogenesis assays showed that host ranges of all three overlap, but are not identical. The host ranges of individual strains of P. carotovorum subsp. carotovorum and subsp. brasiliensis are limited, whereas P. atrosepticum can macerate many plant species in addition to potato. There was high variability in virulence assays with potato tuber; thus physiological factors were investigated. Tuber size, maturity, and field location had significant effects on susceptibility to soft rot, with larger, more mature tubers being more susceptible.

scholarly journals Lipopolysaccharide composition determines the preferred route and entry kinetics of bacterial outer membrane vesicles into host cells

2016 ◽  
Author(s):  
Eloise J O’Donoghue ◽  
Douglas F. Browning ◽  
Ewa Bielska ◽  
Luke Alderwick ◽  
Sara Jabbari ◽  
...  
Keyword(s):  
Chemical Composition ◽  
Outer Membrane ◽  
Real Time ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Gram Negative ◽  
Immune Priming ◽  
Cargo Delivery ◽  
Kinetics Of

SUMMARYOuter membrane vesicles are microvesicles shed by Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle - host cell interactions is limited by a lack of methods to study the kinetics of vesicle entry and cargo delivery to host cells in real-time. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded probe targeted to vesicles. We found that route of vesicular uptake, and thus entry kinetics and efficiency of cargo release, are determined by the chemical composition of the bacterial lipopolysaccharide. The presence of O-antigen facilitates receptor-independent entry, which enhances both rate and efficiency of cargo uptake by host cells. Collectively, our findings highlight the chemical composition of the bacterial cell wall as a major determinant of secretion-independent delivery of virulence factors during Gram-negative infections.

scholarly journals Escherichia coli tol-pal Mutants Form Outer Membrane Vesicles

1998 ◽  
Vol 180 (18) ◽  
pp. 4872-4878 ◽  
Author(s):  
Alain Bernadac ◽  
Marthe Gavioli ◽  
Jean-Claude Lazzaroni ◽  
Satish Raina ◽  
Roland Lloubès
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Membrane Integrity ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Extracellular Medium ◽  
Solid Media ◽  
Outer Membrane Lipoprotein ◽  
Periplasmic Proteins ◽  
Outer Membrane Permeability

ABSTRACT Mutations in the tol-pal genes induce pleiotropic effects such as release of periplasmic proteins into the extracellular medium and hypersensitivity to drugs and detergents. Other outer membrane defective strains such as tolC, lpp, and rfa mutations are also altered in their outer membrane permeability. In this study, electron microscopy and Western blot analyses were used to show that strains with mutations in each of thetol-pal genes formed outer membrane vesicles after growth in standard liquid or solid media. This phenotype was not observed intolC and rfaD cells in the same conditions. AtolA deletion in three different Escherichia coli strains was shown to lead to elevated amounts of vesicles. These results, together with plasmid complementation experiments, indicated that the formation of vesicles resulted from the defect of any of the Tol-Pal proteins. The vesicles contained outer membrane trimeric porins correctly exposed at the cell surface. Pal outer membrane lipoprotein was also immunodetected in the vesicle fraction oftol strains. The results are discussed in view of the role of the Tol-Pal transenvelope proteins in maintaining outer membrane integrity by contributing to target or integrate newly synthesized components of this structure.

scholarly journals Harpin Mediates Cell Aggregation in Erwinia chrysanthemi 3937

2006 ◽  
Vol 188 (6) ◽  
pp. 2280-2284 ◽  
Author(s):  
Yap Mee-Ngan ◽  
Clemencia M. Rojas ◽  
Ching-Hong Yang ◽  
Amy O. Charkowski
Keyword(s):  
Cell Surface ◽  
Hypersensitive Response ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Cell Aggregation ◽  
Soft Rot ◽  
Secretion System ◽  
Erwinia Chrysanthemi ◽  
Type Iii

ABSTRACT The hypersensitive response elicitor harpin (HrpN) of soft rot pathogen Erwinia chrysanthemi strains 3937 and EC16 is secreted via the type III secretion system and remains cell surface bound. Strain 3937 HrpN is essential for cell aggregation, but the C-terminal one-third of the protein is not required for aggregative activity.

scholarly journals Analysis of Erwinia chrysanthemi EC16 pelE∷uidA, pelL∷uidA, and hrpN∷uidA Mutants Reveals Strain-Specific Atypical Regulation of the Hrp Type III Secretion System

2004 ◽  
Vol 17 (2) ◽  
pp. 184-194 ◽  
Author(s):  
Jong Hyun Ham ◽  
Yaya Cui ◽  
James R. Alfano ◽  
Pablo Rodríguez-Palenzuela ◽  
Clemencia M. Rojas ◽  
...  
Keyword(s):  
Growth Phase ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Medium Composition ◽  
Erwinia Chrysanthemi ◽  
Type Ii ◽  
Logarithmic Growth Phase ◽  
Direct Production ◽  
Type Iii

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL∷ΩCmr and hrpS∷ΩCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative σ54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE∷uidA, pelL∷uidA, and hrpN∷uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.

Leishmania LABCG2 transporter is involved in ATP-dependent transport of thiols

2018 ◽  
Vol 475 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Ana Perea ◽  
José Ignacio Manzano ◽  
Yasuhisa Kimura ◽  
Kazumitsu Ueda ◽  
Santiago Castanys ◽  
...  
Keyword(s):  
Green Fluorescence Protein ◽  
Membrane Vesicles ◽  
Protozoan Parasite ◽  
Cell System ◽  
Extracellular Medium ◽  
Redox Metabolism ◽  
Protein Thiols ◽  
Ic50 Value ◽  
Fluorescence Protein ◽  
Dependent Transport

The Leishmania LABCG2 transporter has a key role in the redox metabolism of these protozoan parasites. Recently, the involvement of LABCG2 in virulence, autophagy and oxidative stress has been described. Null mutant parasites for LABCG2 present an increase in the intracellular levels of glutathione (GSH) and trypanothione [T(SH)2]. On the other hand, parasites overexpressing LABCG2 transporter export non-protein thiols to the extracellular medium. To explore if LABCG2 may mediate an active transport of non-protein thiols, the effect of these molecules on ATPase activity of LABCG2 as well as the ability of LABCG2 to transport them was determined using a baculovirus-Sf9 insect cell system. Our results indicate that all thiols tested [GSH, T(SH)2] as well as their oxidized forms GSSG and TS2 (trypanothione disulfide) stimulate LABCG2-ATPase basal activity. We have measured the transport of [3H]-GSH in inside-out Sf9 cell membrane vesicles expressing LABCG2-GFP (green fluorescence protein), finding that LABCG2 was able to mediate a rapid and concentration-dependent uptake of [3H]-GSH in the presence of ATP. Finally, we have analyzed the ability of different thiol species to compete for this uptake, T(SH)2 and TS2 being the best competitors. The IC50 value for [3H]-GSH uptake in the presence of increasing concentrations of T(SH)2 was less than 100 μM, highlighting the affinity of this thiol for LABCG2. These results provide the first direct evidence that LABCG2 is an ABC transporter of reduced and oxidized non-protein thiols in Leishmania, suggesting that this transporter can play a role in the redox metabolism and related processes in this protozoan parasite.

Sign in / Sign up

Export Citation Format

Share Document