Membrane vesicles, nanopods and/or nanotubes produced by hyperthermophilic archaea of the genus Thermococcus

2013 ◽  
Vol 41 (1) ◽  
pp. 436-442 ◽  
Author(s):  
Evelyne Marguet ◽  
Marie Gaudin ◽  
Emilie Gauliard ◽  
Isabelle Fourquaux ◽  
Stephane le Blond du Plouy ◽  
...  
Keyword(s):  
Cellular Networks ◽  
Culture Medium ◽  
Cell Envelope ◽  
Protein A ◽  
Peptide Binding ◽  
Membrane Vesicles ◽  
Hyperthermophilic Archaea ◽  
Eukaryotic Cells ◽  
Major Protein ◽  
Tubular Structures

Thermococcus species produce MVs (membrane vesicles) into their culture medium. These MVs are formed by a budding process from the cell envelope, similar to ectosome formation in eukaryotic cells. The major protein present in MVs of Thermococci is a peptide-binding receptor of the OppA (oligopeptide-binding protein A) family. In addition, some of them contain a homologue of stomatin, a universal membrane protein involved in vesiculation. MVs produced by Thermococcus species can recruit endogenous or exogenous plasmids and plasmid transfer through MVs has been demonstrated in Thermococcus kodakaraensis. MVs are frequently secreted in clusters surrounded by S-layer, producing either big protuberances (nanosphere) or tubular structures (nanotubes). Thermococcus gammatolerans and T. kodakaraensis produce nanotubes containing strings of MVs, resembling the recently described nanopods in bacteria, whereas Thermococcus sp. 5-4 produces filaments whose internal membrane is continuous. These nanotubes can bridge neighbouring cells, forming cellular networks somehow resembling nanotubes recently observed in Firmicutes. As suggested for bacteria, archaeal nanopods and/or nanotubes could be used to expand the metabolic sphere around cells and/or to promote intercellular communication.

scholarly journals The peptidoglycan-associated protein NapA plays an important role in the envelope integrity and in the pathogenesis of the lyme disease spirochete

2021 ◽  
Vol 17 (5) ◽  
pp. e1009546
Author(s):  
Marisela M. Davis ◽  
Aaron M. Brock ◽  
Tanner G. DeHart ◽  
Brittany P. Boribong ◽  
Katherine Lee ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Outer Membrane ◽  
Structural Integrity ◽  
Molecular Beacon ◽  
Cell Envelope ◽  
Protein A ◽  
Bacterial Pathogen ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Gram Negative

The bacterial pathogen responsible for causing Lyme disease, Borrelia burgdorferi, is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. In diderms, peptidoglycan (PG) is sandwiched between the inner and outer membrane of the cell envelope. In many other Gram-negative bacteria, PG is bound by protein(s), which provide both structural integrity and continuity between envelope layers. Here, we present evidence of a peptidoglycan-associated protein (PAP) in B. burgdorferi. Using an unbiased proteomics approach, we identified Neutrophil Attracting Protein A (NapA) as a PAP. Interestingly, NapA is a Dps homologue, which typically functions to bind and protect cellular DNA from damage during times of stress. While B. burgdorferi NapA is known to be involved in the oxidative stress response, it lacks the critical residues necessary for DNA binding. Biochemical and cellular studies demonstrate that NapA is localized to the B. burgdorferi periplasm and is indeed a PAP. Cryo-electron microscopy indicates that mutant bacteria, unable to produce NapA, have structural abnormalities. Defects in cell-wall integrity impact growth rate and cause the napA mutant to be more susceptible to osmotic and PG-specific stresses. NapA-linked PG is secreted in outer membrane vesicles and augments IL-17 production, relative to PG alone. Using microfluidics, we demonstrate that NapA acts as a molecular beacon—exacerbating the pathogenic properties of B. burgdorferi PG. These studies further our understanding of the B. burgdorferi cell envelope, provide critical information that underlies its pathogenesis, and highlight how a highly conserved bacterial protein can evolve mechanistically, while maintaining biological function.

Expression and transport of rabbit surfactant protein A in COS-1 cells

1992 ◽  
Vol 262 (4) ◽  
pp. L437-L445 ◽  
Author(s):  
J. L. Alcorn ◽  
Q. Chen ◽  
V. Boggaram ◽  
C. R. Mendelson
Keyword(s):  
Culture Medium ◽  
Lung Surfactant ◽  
Protein A ◽  
Apparent Molecular Weight ◽  
Subcellular Fractionation ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Major Protein ◽  
Rabbit Lung ◽  
Transfected Cells

SV40-transformed green monkey kidney (COS-1) cells were transfected with expression plasmids that contained either the structural gene or cDNA for surfactant protein A (SP-A), a major protein of rabbit lung surfactant. The transfected COS-1 cells synthesized several isoforms of SP-A that were found to be less acidic than those produced in rabbit lung tissue. SP-A species with apparent molecular weight (M(r)) approximately equal to 29,000–33,000 were detected in the transfected cells, whereas glycosylated forms with apparent M(r) approximately equal to 33,000–38,000 were detectable only in the culture medium. Analysis of transfected cells by indirect immunofluorescence revealed that SP-A was localized in punctate bodies throughout the cytoplasm. Expressed SP-A was not detectable on the cell surface nor was there evidence that secreted SP-A was endocytosed by COS-1 cells. After subcellular fractionation of the transfected COS-1 cells, SP-A was found to be localized predominantly in the 5,000- and 18,000-g pellet fractions; little or no immunoreactive SP-A was detectable in cytosolic fractions. Treatment of transfected cells with the glycosylation inhibitor tunicamycin prevented secretion of SP-A into the medium, suggesting a role of glycosylation in secretion of SP-A. On the other hand, treatment of transfected cells with inhibitors of proline hydroxylation, which may cause destabilization of the collagen-like domain of SP-A, reduced but did not prevent secretion of SP-A into the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Genetic Analysis of Factors Affecting Susceptibility of Bacillus subtilis to Daptomycin

2009 ◽  
Vol 53 (4) ◽  
pp. 1598-1609 ◽  
Author(s):  
Anna-Barbara Hachmann ◽  
Esther R. Angert ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Transcriptional Profiling ◽  
Protein A ◽  
Multidrug Resistant ◽  
Dependent Manner ◽  
Membrane Composition ◽  
Factors Affecting ◽  
Anionic Phospholipids ◽  
Cyclic Lipopeptide

ABSTRACT Daptomycin is the first of a new class of cyclic lipopeptide antibiotics used against multidrug-resistant, gram-positive pathogens. The proposed mechanism of action involves disruption of the functional integrity of the bacterial membrane in a Ca2+-dependent manner. We have used transcriptional profiling to demonstrate that treatment of Bacillus subtilis with daptomycin strongly induces the lia operon including the autoregulatory LiaRS two-component system (homologous to Staphylococcus aureus VraSR). The lia operon protects against daptomycin, and deletion of liaH, encoding a phage-shock protein A (PspA)-like protein, leads to threefold increased susceptibility. Since daptomycin interacts with the membrane, we tested mutants with altered membrane composition for effects on susceptibility. Deletion mutations of mprF (lacking lysyl-phosphatidylglycerol) or des (lipid desaturase) increased daptomycin susceptibility, whereas overexpression of MprF decreased susceptibility. Conversely, depletion of the cell for the anionic lipid phosphatidylglycerol led to increased resistance. Fluorescently labeled daptomycin localized to the septa and in a helical pattern around the cell envelope and was delocalized upon the depletion of phosphatidylglycerol. Together, these results indicate that the daptomycin-Ca2+ complex interacts preferentially with regions enriched in anionic phospholipids and leads to membrane stresses that can be ameliorated by PspA family proteins.

scholarly journals Type VI secretion system sheaths as nanoparticles for antigen display

2016 ◽  
Vol 113 (11) ◽  
pp. 3042-3047 ◽  
Author(s):  
Elena Del Tordello ◽  
Olga Danilchanka ◽  
Andrew J. McCluskey ◽  
John J. Mekalanos
Keyword(s):  
Protein A ◽  
Secretion System ◽  
Factor H ◽  
Soluble Antigen ◽  
Tubular Structures ◽  
Type Vi Secretion System ◽  
Interacting Protein ◽  
Serum Bactericidal Assay ◽  
Heterologous Antigens ◽  
Bacterial Type

The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between cells by a mechanism analogous to phage tail contraction. T6SS sheaths are cytoplasmic tubular structures composed of stable VipA-VipB (named for ClpV-interacting protein A and B) heterodimers. Here, the structure of the VipA/B sheath was exploited to generate immunogenic multivalent particles for vaccine delivery. Sheaths composed of VipB and VipA fused to an antigen of interest were purified from Vibrio cholerae or Escherichia coli and used for immunization. Sheaths displaying heterologous antigens generated better immune responses against the antigen and different IgG subclasses compared with soluble antigen alone. Moreover, antigen-specific antibodies raised against sheaths presenting Neisseria meningitidis factor H binding protein (fHbp) antigen were functional in a serum bactericidal assay. Our results demonstrate that multivalent nanoparticles based on the T6SS sheath represent a versatile scaffold for vaccine applications.

The outer membrane of Haemophilus influenzae type b: cell envelope associations of major proteins

1983 ◽  
Vol 29 (2) ◽  
pp. 280-287 ◽  
Author(s):  
J. W. Coulton ◽  
D. T. F. Wan
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Haemophilus Influenzae Type ◽  
Major Protein ◽  
Haemophilus Influenzae Type B ◽  
Type B ◽  
Molecular Weights ◽  
Triton X

Membrane proteins fom the cell envelope of Haemophilus influenzae type b ATCC 9795 were examined by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. When envelopes were extracted with a phosphate-based buffer containing 2% Triton X-100, a major protein of molecular weight 43 000 was detected in fractions containing cytoplasmic membrane proteins. The cell wall material which was Triton X-100 insoluble contained six major proteins of molecular weights 46 000, 40 000, 36 000, 30 000, 27 000, and 16 000. One of these proteins showed a shift in molecular weight from 27 000 to 36 000 when it was heated over a temperature range from 50 °C to 100 °C in buffer containing 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol. This alteration in mobility could be demonstrated either by the membrane-bound form of the protein or by a detergent-soluble form of the protein. Enriched preparations of the 36 000 molecular weight form were obtained by a series of purification steps. Extraction of the Triton X-100 insoluble material with buffer containing 2% Triton X-100, 5.0 mM EDTA yielded chiefly one major protein molecular weight 30 000 and many minor protein species. Pretreatment of the Triton X-100 insoluble fraction with lysozyme followed by extraction with buffer containing 2% Triton X-100, 5.0 mM EDTA released two proteins of molecular weights 16 000 and 27 000 and few minor proteins. By these operational manipulations, the proteins of molecular weights 16 000 and 27 000 may be considered as peptidoglycan-associated proteins.

scholarly journals Antibiotic action revealed by real-time imaging of the mycobacterial membrane

2022 ◽  
Author(s):  
Michael G. Wuo ◽  
Charles L Dulberger ◽  
Robert A. Brown ◽  
Alexander Sturm ◽  
Eveline Ultee ◽  
...  
Keyword(s):  
Real Time ◽  
Immune Modulation ◽  
Cell Envelope ◽  
Membrane Vesicles ◽  
Time Lapse ◽  
Outer Membrane Vesicles ◽  
Imaging Data ◽  
Minimal Cell ◽  
Mycobacterial Cell ◽  
Cellular Phenotypes

The current understanding of mycobacterial cell envelope remodeling in response to antibiotics is limited. Chemical tools that report on phenotypic changes with minimal cell wall perturbation are critical to gaining insight into this time-dependent phenomenon. Herein we describe a fluorogenic chemical probe that reports on mycobacterial cell envelope assembly in real time. We used time-lapse microscopy to reveal distinct spatial and temporal changes in the mycobacterial membrane upon treatment with frontline antibiotics. Differential antibiotic treatment elicited unique cellular phenotypes, providing a platform for monitoring cell envelope construction and remodeling responses simultaneously. Analysis of the imaging data indicates a role for antibiotic-derived outer membrane vesicles in immune modulation.

scholarly journals Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins

2020 ◽  
Vol 21 (15) ◽  
pp. 5496
Author(s):  
Claire Siebert ◽  
Corinne Mercier ◽  
Donald K. Martin ◽  
Patricia Renesto ◽  
Beatrice Schaack
Keyword(s):  
Outer Membrane ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Iron Transport ◽  
Molecular Mechanisms ◽  
Protein A ◽  
Membrane Vesicles ◽  
Disodium Salt ◽  
Outer Membrane Vesicles ◽  
Novel Therapeutic Strategies

Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections.

scholarly journals The Mutation of Conservative Asp268 Residue in the Peptidoglycan-Associated Domain of the OmpA Protein Affects Multiple Acinetobacter baumannii Virulence Characteristics

Molecules ◽  
2019 ◽  
Vol 24 (10) ◽  
pp. 1972 ◽  
Author(s):  
Jūratė Skerniškytė ◽  
Emilija Karazijaitė ◽  
Julien Deschamps ◽  
Renatas Krasauskas ◽  
Romain Briandet ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Protein A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Clinical Strain ◽  
Infection Model ◽  
Terminal Domain ◽  
Ompa Protein

Acinetobacter baumannii is a nosocomial human pathogen of increasing concern due to its multidrug resistance profile. The outer membrane protein A (OmpA) is an abundant bacterial cell surface component involved in A. baumannii pathogenesis. It has been shown that the C-terminal domain of OmpA is located in the periplasm and non-covalently associates with the peptidoglycan layer via two conserved amino acids, thereby anchoring OmpA to the cell wall. Here, we investigated the role of one of the respective residues, D268 in OmpA of A. baumannii clinical strain Ab169, on its virulence characteristics by complementing the ΔompA mutant with the plasmid-borne ompAD268A allele. We show that while restoring the impaired biofilm formation of the ΔompA strain, the Ab169ompAD268A mutant tended to form bacterial filaments, indicating the abnormalities in cell division. Moreover, the Ab169 OmpA D268-mediated association to peptidoglycan was required for the manifestation of twitching motility, desiccation resistance, serum-induced killing, adhesion to epithelial cells and virulence in a nematode infection model, although it was dispensable for the uptake of β-lactam antibiotics by outer membrane vesicles. Overall, the results of this study demonstrate that the OmpA C-terminal domain-mediated association to peptidoglycan is critical for a number of virulent properties displayed by A. baumannii outside and within the host.

scholarly journals Cysteine Is Exported from theEscherichia coliCytoplasm by CydDC, an ATP-binding Cassette-type Transporter Required for Cytochrome Assembly

2002 ◽  
Vol 277 (51) ◽  
pp. 49841-49849 ◽  
Author(s):  
Marc S. Pittman ◽  
Hazel Corker ◽  
Guanghui Wu ◽  
Marie B. Binet ◽  
Arthur J. G. Moir ◽  
...  
Keyword(s):  
Redox Homeostasis ◽  
Membrane Vesicles ◽  
Major Facilitator Superfamily ◽  
Atp Binding ◽  
Major Protein ◽  
Wild Type ◽  
Independent Manner ◽  
Periplasmic Transport ◽  
Major Facilitator ◽  
Atp Binding Cassette

Assembly ofEscherichia colicytochromebdand periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type andcydDmutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in thecydDperiplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropiccydDphenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmicc-type cytochromes incydDand wild-type strains but did not restore cytochromed. Consistent with CydDC being a cysteine exporter,cydDmutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective inorf299, encoding a transporter of the major facilitator superfamily. AcydD orf299double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.

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