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PubMed2025

TamL is a Key Player of the Outer Membrane Homeostasis in Bacteroidota.

In Proteobacteria, the outer membrane protein TamA and the inner membrane-anchored protein TamB form the Translocation and Assembly Module (TAM) complex, which facilitates the transport of autotransporters, virulence factors, and likely lipids across the two membranes. In Bacteroidota, TamA is replaced by TamL, a TamA-like lipoprotein with a lipid modification at its N-terminus that likely anchors it to the outer membrane. This structural difference suggests that TamL may have a distinct function compared to TamA. However, the role of TAM in bacterial phyla other than Proteobacteria remains unexplored. Our study aimed to elucidate the function of TamL in Flavobacterium johnsoniae, an environmental Bacteroidota. Unlike its homologs in Proteobacteria, we found that TamL and TamB are essential in F. johnsoniae. Through genetic, phenotypic, proteomic, and lipidomic analyses, we show that TamL depletion severely compromises outer membrane integrity, as evidenced by reduced cell viability, altered cell shape, increased susceptibility to membrane-disrupting agents, and elevated levels of outer membrane lipoproteins. Notably, we did not observe an overall decrease in the levels of β-barrel outer membrane proteins, nor substantial alterations in outer membrane lipid composition. By pull-down assays, we found TamL co-purifying with TamB in F. johnsoniae, suggesting an interaction. Furthermore, we found that while TamL and TamB monocistronic genes are conserved among Bacteroidota, only some species encode multiple TamL, TamB and TamA proteins. To our knowledge, this study is the first to provide functional insights into a TAM subunit beyond Proteobacteria.

Bacterial Outer Membrane Proteins↗
PubMed1998

Modulation of interleukin-8 activity by gingipains from Porphyromonas gingivalis: implications for pathogenicity of periodontal disease.

Gingipains are the major cysteine proteinases synthesized by Porphyromonas gingivalis which, in soluble form, are able to initially convert IL-8 (77 amino acid residues) to a more potent species truncated at the amino terminus, followed by slow degradation and destruction of chemokine biological activity. In contrast, the same enzymes when associated with bacterial outer-membrane blebs (vesicles), instantly degrade this chemokine. This division of enhancing and inactivating activity between soluble and membrane-bound gingipains can cause the compartmentalization of pro- and anti-inflammatory reactions to distal and proximal positions from bacterial plaque, respectively, which may explain why, despite the massive neutrophil accumulation at periodontitis sites, there is no elimination of infection.

Adhesins, Bacterial↗
PubMed1997

Complex inhibition of OmpF and OmpC bacterial porins by polyamines.

The effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on the activity of bacterial porins OmpC and OmpF were investigated by electrophysiology. Membrane vesicles made from the outer membrane of Escherichia coli strains expressing only OmpC or OmpF were reconstituted into liposomes probed by patch clamp. The channel activity was recorded in control solutions and in the presence of increasing concentrations of a specific polyamine. In all cases, concentration- and voltage-dependent inhibitory effects were observed. They include both the suppression of channel openings and the enhancement of channel closures as well as the promotion of blocked or inactivated states. OmpF and OmpC, although highly homologous, have distinct sensitivities to modulation, especially by spermine. This compound inhibits OmpF in the nanomolar range, which is in agreement with its potency on eukaryotic channels. Putrescine was the least effective (upper millimolar range) and also had inhibitory effects qualitatively distinct from those exerted by the other polyamines. The compounds appear to bind to at least two distinct binding sites, one of which resides within the pore. The potencies to this site are lower when the polyamines are applied from the extracellular side than from the periplasmic side, suggesting an asymmetric binding site.

Binding Sites↗
PubMed1999

Virulence factors of Actinobacillus actinomycetemcomitans.

A. actinomycetemcomitans has clearly adapted well to its environs; its armamentarium of virulence factors (Table 2) ensures its survival in the oral cavity and enables it to promote disease. Factors that promote A. actinomycetemcomitans colonization and persistence in the oral cavity include adhesins, bacteriocins, invasins and antibiotic resistance. It can interact with and adhere to all components of the oral cavity (the tooth surface, other oral bacteria, epithelial cells or the extracellular matrix). The adherence is mediated by a number of distinct adhesins that are elements of the cell surface (outer membrane proteins, vesicles, fimbriae or amorphous material). A. actinomycetemcomitans enhances its chance of colonization by producing actinobacillin, an antibiotic that is active against both streptococci and Actinomyces, primary colonizers of the tooth surface. The fact that A. actinomycetemcomitans resistance to tetracyclines, a drug often used in the treatment of periodontal disease, is on the rise is an added weapon. Periodontal pathogens or their pathogenic products must be able to pass through the epithelial cell barrier in order to reach and cause destruction to underlying tissues (the gingiva, cementum, periodontal ligament and alveolar bone). A. actinomycetemcomitans is able to elicit its own uptake into epithelial cells and its spread to adjacent cells by usurping normal epithelial cell function. A. actinomycetemcomitans may utilize these remarkable mechanisms for host cell infection and migration to deeper tissues. A. actinomycetemcomitans also orchestrates its own survival by elaborating factors that interfere with the host's defense system (such as factors that kill phagocytes and impair lymphocyte activity, inhibit phagocytosis and phagocyte chemotaxis or interfere with antibody production). Once the organisms are firmly established in the gingiva, the host responds to the bacterial onslaught, especially to the bacterial lipopolysaccharide, by a marked and continual inflammatory response, which results in the destruction of the periodontal tissues. A. actinomycetemcomitans has at least three individual factors that cause bone resorption (lipopolysaccharide, proteolysis-sensitive factor and GroEL), as well as a number of activities (collagenase, fibroblast cytotoxin, etc.) that elicit detrimental effects on connective tissue and the extracellular matrix. It is of considerable interest to know that A. actinomycetemcomitans possesses so many virulence factors but unfortunate that only a few have been extensively studied. If we hope to understand and eradicate this pathogen, it is critical that in-depth investigations into the biochemistry, genetic expression, regulation and mechanisms of action of these factors be initiated.

Abscess↗
PubMed1995

Characteristics of Actinobacillus actinomycetemcomitans invasion of and adhesion to cultured epithelial cells.

Actinobacillus actinomycetemcomitans (A.a.) is highly implicated in periodontitis. We have developed several in vitro models using the KB oral cell line to examine A.a.-epithelial cell interactions. In support of the use of KB cell line model systems is our finding that A.a. invaded KB and primary gingival cells to the same extent. Invasion is an active event which requires new protein synthesis by both KB and A.a. Like many other intracellular parasites, A.a. invade by receptor-mediated endocytosis. We observed that internalized A.a. were surrounded by foci of actin which had been transported from the periphery of the KB cell. Adhesion of A.a. to KB cells occurred rapidly and stimulated the formation of microvilli. Adhesion is affected by both host factors (saliva, serum, [NaCl]) and culture conditions. Multiple determinants [fimbriae, outer membrane proteins, vesicles, and/or an extracellular amorphous material (ExAmMat)] which are either associated with the A.a. surface or are released into the milieu are involved. We determined that ExAmMat can convey adhesiveness to weakly adherent A.a. and to at least one other oral species (Streptococcus parasanguis).

Adhesins, Bacterial↗
PubMed1983

Cellular immunotoxicity of amyl nitrite.

Functional deficits in lymphocyte interaction following occasional or chronic exposure to inhaled nitrites may be a potential contributing but not the etiologic factor in the acquired immunodeficiency syndrome (AIDS). We evaluated the effect of amyl nitrite vapors on mononuclear cell function and demonstrated functional deficits and structural alterations in these cells. In this closed, in vitro system, exposure of cells to amyl nitrite for up to 30 minutes did not effect cell viability. The functional deficits demonstrated were: inhibition of lymphocyte erythrocyte (E) rosette formation, a suppression of lymphocyte mitogen (phytohemagglutinin) and antigen (cytomegalovirus) transformation, a block in the S, G2 and M phases of cell cycling and diminished cell cytotoxicity to CMV infected cells. These effects on cellular function were demonstrated following 5, 10, and 15 minutes of amyl nitrite vapor exposure; some effect on all cellular functions was demonstrated at 5 minutes. The structural alterations seen on scanning and transmission electron micrographs were: reduction of filapodia, smoothing of the cell profile, cytoplasmic protrusions with pseudopod-like extensions, an increase in rough endoplasmic reticulum with swollen cisternae, alterations in size and distribution of golgi components and exocytotic vesicles in the outer membrane of the nuclear envelope. These vesicles and increased membrane proliferation suggests an effect on the membrane synthesis mechanism in these cells. These effects may be a potential factor in the alterations of phenotypic markers on T lymphocyte populations, as well as, a potential contributing factor in the functional deficit of mononuclear cells in patients with AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1978

[Mitochondrial localization of cytochrome c1 with specific antibodies].

A specific antibody against cytochrome c1 (pig heart mitochondria) has been obtained. It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome c1 site of inner mitochondrial membrane ; but it has no effect on the isolated submitochondrial particles (inside-out inner mitochondrial membrane vesicles free of any outer membrane or outside-out inner membrane). Thus the topologic position of cytochrome c1 in the inner mitochondrial membrane is asymetrically lcoated on the outer side of the inner mitochondrial membrane. These results agree with our previous researches on ATP-ase and cytochromes b, c and a, indicating the location on the inner side for the first one, transmembranous for the last one, on the outer side for the others respiratory chain components. Thus the electron transport from cytochrome b to a takes place in the outer region of inner mitochondrial membrane and the transmembranous location of cytochrome-oxidase facilitates the transfer of the electrons to oxygen.

Animals↗
PubMed1984

Stable insertion of C5b-9 complement complexes into the outer membrane of serum treated, susceptible Escherichia coli cells as a prerequisite for killing.

Escherichia coli 17, a K12 derivative, was rapidly killed by human serum following a short lag period of 10 min. Stable binding of terminal C5b-9 complement complexes was investigated in time course experiments. Serum treated E. coli cells were lysed osmotically and the resulting outer and cytoplasmic membrane vesicles separated by sucrose gradient centrifugation. Exposure of E. coli 17 to serum rapidly reduced the degree of recoverability of cytoplasmic membrane vesicles. Electron microscopy revealed no interaction of C5b-9 complexes with CM vesicles. In contrast there was a clear time-dependent deposition of terminal complement complexes onto OM-vesicles. Very few complexes were detected during the prekilling phase of the reaction; initiation of the active killing phase was accompanied by a large increase in complement lesions. In contrast, no C5b-9 complexes could be visualised on outer or cytoplasmic membrane vesicles of a smooth, serum-resistant E. coli strain. We conclude that complement-mediated killing is a consequence of stable binding of C5b-9 complexes to the outer membrane of susceptible strains.

Blood Bactericidal Activity↗
PubMed1993

Localization of the meningococcal receptors for human transferrin.

The interaction between gold-labelled human transferrin (Au-HTF) with live meningococci after growth in vivo or in different in vitro conditions was examined by electron microscopy to localize and quantify the numbers of HTF-binding sites on the cell surface. It was clearly demonstrated that HTF binds to the surface of live meningococci (of different serogroups and serotypes) after growth in either iron-sufficient or iron-restricted cultures, although the degree of labelling was always higher (2- to 35-fold) in the latter case. The commensal Neisseria polysaccharea behaved similarly. Ultrathin sections showed that Au-HTF was localized predominantly on the outer membrane of the cells and vesicles, with hardly any internalization. Au-HTF labelling on meningococci was significantly reduced after incubation with unlabelled HTF or with rabbit antiserum containing antibodies against transferrin-binding proteins (TBPs), demonstrating the specificity of the interaction. These sera also blocked binding between HTF and outer membrane proteins on Western immunoblots. Direct evidence of the expression of the TBPs (Western blots) and localization of the HTF receptor (electron microscopy) on in vivo-grown meningococci was obtained from organisms derived without laboratory culturing from the cerebrospinal fluid of a patient. There was considerable cell-to-cell variation in the amount of labelling present on cells of the same sample (in vitro- or in vivo-grown organisms) and between different strains. The degree of binding varied with time of incubation of the cells with Au-HTF. The gold particles frequently formed discrete circles on the cell surfaces of the in vitro-grown organisms; these circles appear to be associated with outer membrane vesicle formation. The results show that the TBPs, which form part of the active components of the HTF receptor(s), are expressed in vivo and are surface exposed and immunogenic and that antibodies against them can interfere with the HTF binding of the meningococcal cells, which may affect iron utilization. This study further supports the concept of regarding the TBPs as future vaccine candidates.

Animals↗
PubMed1995

Intimate association of H(+)-ATPase vesicles with mitochondria in the intercalated cell of the renal collecting duct.

It is known that the intercalated cell of the renal collecting duct contains, in addition to abundant mitochondria, characteristic spherical or flat vesicles whose cytoplasmic surfaces were decorated with H(+)-ATPase studs. The intimate association of H(+)-ATPase vesicles with mitochondria was often observed. Here, the limiting membrane of the vesicle and the outer mitochondrial membrane were directly connected to each other by H(+)-ATPase studs. Namely, two membranes were bridged by these studs. This feature was especially evident in the case of flat vesicles. These findings indicated the close interplay between these two cell organellae.

Animals↗
PubMed1992

Role of Porphyromonas gingivalis 40-kDa outer membrane protein in the aggregation of P. gingivalis vesicles and Actinomyces viscosus.

Porphyromonas gingivalis, an important pathogen in periodontitis, produces extracellular vesicles that aggregate with Actinomyces viscosus cells. A 40-kDa outer membrane protein (OMP)-coding gene from P. gingivalis was cloned and the protein was found to be localized in these vesicles. The recombinant 40-kDa OMP did not show aggregation activity. However, affinity-purified antibody against the recombinant protein significantly inhibited aggregation of P. gingivalis vesicles with A. viscosus cells. The antibody also inhibited cellular coaggregation of several strains of P. gingivalis with A. viscosus cells, but not with other periodontal pathogens. Moreover, aggregation of A. viscosus cells with P. gingivalis vesicles was inhibited in a dose-dependent manner by pre-treatment of the A. viscosus cells with the recombinant protein. These findings suggest that the 40-kDa OMP may be an important aggregation factor of P. gingivalis.

Actinomyces viscosus↗
PubMed1998

Novel fluorescence membrane fusion assays reveal GTP-dependent fusogenic properties of outer mitochondrial membrane-derived proteins.

We have shown that fusion of small unilamellar vesicles (SUV) with outer mitochondrial membranes occurs at physiological pH [Cortese et al., 1991, J. Cell Biol., Vol. 113, 1331-1340]. The proteins driving this process could be involved in mitochondrial membrane fusion, which is presently poorly understood. In this study, we release from rat liver mitochondria a soluble protein fraction (SF) that increases fusion at neutral pH measured by membrane fusion assays (MFAs). Since this fusogenic activity was specifically enhanced by GTP, we separate SF by GTP affinity chromatography into: i) a flow-through subfraction (G1) containing numerous proteins with low GTP affinity; and ii) a subfraction (G2) which may contain GTP-binding proteins. A novel array of MFAs is developed to study the fusogenic properties of these fractions, measuring the merging of membranes (membrane-mixing) or the mixing of intravesicular aqueous contents (content-mixing). The MFAs use: a) SUV/large unilamellar vesicles, lacking mitochondrial membranes; b) SUV/mitochondria, reconstituting membrane-mitochondrial interactions; and c) mitochondria/mitochondria, mimicking mitochondrial fusion. The results indicate that: i) G1 contains GTP-independent, in vitro fusogenic proteins that are not sufficient to induce mitochondrial fusion; and ii) G2 contains GTP-dependent proteins that stimulate mitochondrial fusion at neutral pH. The MFAs described here could be used to monitor the isolation of active proteins from these subfractions and to define the mechanism of intermitochondrial membrane fusion.

Animals↗
PubMed1997

Cell lines susceptible to infection are permeabilized by cleaved and solubilized outer layer proteins of rotavirus.

It has previously been shown that trypsinized triple-layered particles of rotavirus induce destabilization of liposomes and membrane vesicles in the absence of Ca2+, a condition which leads to solubilization of the outer capsid proteins of the virus. In this work, we have studied the relationship between outer capsid solubilization and permeabilization of membrane vesicles, monitoring particle and vesicle size simultaneously by changes in light scattering. Permeabilization of intact cells induced by solubilized outer capsid proteins was monitored by following the rate of entry of ethidium bromide into the cells. Solubilized outer capsid proteins separated from double-layered particles induced vesicle permeabilization. Solubilization of the outer capsid preceded and was required for vesicle or cell permeabilization. Membrane damage induced by rotaviral outer proteins was not repaired upon addition of 1 mM Ca2+ to the medium. Rotavirus infection and cell permeabilization were correlated in six different cell lines tested. This phenomenon might be related to the mechanism of virus entry into the cell. We propose a new model for rotavirus internalization based on the permeabilizing ability of outer capsid proteins and the cycling of trapped calcium in the endosomal compartment.

Capsid↗
PubMed1983

Alteration of the immunoglobulin G subclass responses in mice to lipopolysaccharide: effects of nonbacterial proteins and bacterial membrane phospholipids or outer membrane proteins of Proteus mirabilis.

The immunoglobulin M (IgM) and the IgG1, IgG2ab, and IgG3 subclasses of plaque-forming cells (PFC) specific for lipopolysaccharide (LPS) were measured after immunization of mice with LPS alone and compared with the responses to LPS in combination with nonbacterial proteins and with bacterial membrane phospholipid vesicles or two major outer membrane proteins from Proteus mirabilis. The relative numbers of IgG PFC belonging to the IgG1, IgG2, or IgG3 subclasses induced by immunization with LPS alone depended upon the type of LPS administered. Phospholipids and the proteins effected characteristic alterations in not only the strength but also the subclass of the IgG responses to LPS. The results suggest that the hydrophobic-hydrophilic nature or state of aggregation of the preparations plays a role in the induction of IgG1 and IgG2 subclasses of PFC specific for LPS. Complex formation with LPS and adjuvant was apparently necessary to obtain these effects.

Adjuvants, Immunologic↗
PubMed1996

Lectin binding patterns in two cultured endothelial cell types derived from bovine corpus luteum.

Epithelial cells of different phenotypes derived from bovine corpus luteum have been studied intensively in our laboratory. In this study, specific lectin binding was examined for cells of type 1 and 3, which were defined as endothelial cells. In order to confirm differences in their glycocalyx at the light microscopic level, five biotinylated lectins were applied to postconfluent cultures which had been fixed with buffered paraformaldehyde or glutaraldehyde. Cells were not permeabilized with any detergent. Lectin binding was localized with a streptavidin-peroxidase complex which was visualized with two different techniques. The DAB technique detected peroxidase histochemically, while the immunogold technique used an anti-peroxidase gold complex together with silver amplification. Neither cell type 1 nor cell type 3 bound a particular lectin selectively, yet each cell type expressed a particular lectin binding pattern. With the DAB technique, diverse lectin binding patterns were seen, probably indicating either "outside" binding, i.e., a diffuse pattern, a lateral-cell-side pattern and a microvillus-like pattern, or "inside" binding, i.e., a diffuse pattern, and a granule-like pattern. With the immunogold technique, only "outside" binding was observed. In addition, the patterns of single cilia or of single circles were detected, the latter roughly representing 3-micron-sized binding sites for concanavalin A. When localizing them at the ultrastructural level, single circles corresponded with micron-sized discontinuities of the plasma membrane. Shedding vesicles were detected whose outer membrane was labelled with concanavalin A. Our results confirm the diversity of the two cell types under study. The "inside" lectin binding may be caused by way of transient plasma membrane openings and related to shedding of right-side out vesicles ("ectocytosis").

Animals↗
PubMed1988

Ultrastructural studies of the early events of the human sperm acrosome reaction as initiated by human follicular fluid.

It has been suggested that the morphology of the human sperm acrosome reaction (AR) is markedly different from that of other mammalian sperm. The present study examines the fine structural events of the early stages of the human sperm AR as initiated by preovulatory human follicular fluid. Human sperm, capacitated in vitro for 6 hr at 40 degrees C (90% motility) were diluted with equal volumes of follicular fluid before fixing in cacodylate-buffered glutaraldehyde at 5, 10, 15, 20, and 180 sec. Fixed sperm were treated with either tannic acid or thiocarbohydrazine and OsO4. Over 2,000 sperm were viewed. By 5 sec, 2% of the sperm had initiated the AR. By 15 sec, 8% of the sperm were in some stage of the reaction, and after 180 sec 40% of the sperm had completed the acrosome reaction. The initial stages of the human AR are characterized by a swelling or decondensation of the acrosomal matrix. The fusion between the plasma membrane and outer acrosomal membrane begins at the most anterior tip of the head and progresses toward the equatorial segment. Fusion and fenestration ended at the equatorial segment. With thiocarbohydrazine + OsO4 fixation the fused membranes are distinct hybrid vesicles with the outer acrosomal membrane being far more electron dense. The acrosomal matrix is retained by 20 sec, but by 180 sec the matrix is completely dispersed, even when viewed after tannic acid fixation. Also by 180 sec, vesicles were being progressively lost. It is therefore concluded that the morphology of the human sperm AR, as initiated by human follicular fluid, is not unique, but is similar to that described for other mammalian sperm.

Acrosome↗
PubMed1979

Direct evidence for formation of hybrid vesicles by fusion of plasma and outer acrosomal membranes during the acrosome reaction in boar spermatozoa.

A variety of treatment procedures was utilized to identify the origin and composition of the vesicles formed during the acrosome reaction of boar spermatozoa. Whether the acrosome reaction occurred spontaneously or was induced chemically the vesicles were hybrid vesicles composed of roughly equal proportions of plasma and outer acrosomal membranes.

Acrosome↗
PubMed2004

Vesicles with a double bilayer.

A modified reverse phase evaporation method was used to prepare intermediate unilamellar vesicles coated with an additional membrane, or large vesicles in which several vesicles were coated with a common membrane. In both kinds of vesicle, the outer and inner membranes are usually of different phospholipid composition. The preparation involves the formation of a double emulsion: vesicles in a buffer are emerged in a low-boiling point organic solution of phospholipids. Then the organic solvent is evaporated during the heating and mixing process. As result large unilamellar vesicles (LUVs), about 100 nm in diameter, were coated with an additional membrane from egg lecithin or dipalmitoylphosphatidylcholine and cholesterol. The highest yield of the coating was about 50%. When DPPC was used for coating above the phase transition temperature Tm, the data suggested the formation of vesicles that were slightly larger than the starting LUVs. It might be concluded that many of these had a double bilayer. If the coating was done below Tm, the micrographs suggested the formation of structures resembling multi-vesicular vesicles. They looked like LUV clusters coated with a common membrane.

Cytoplasmic Vesicles↗