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PubMed2005

Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore.

Tom40 is the central pore-forming component of the translocase of the outer mitochondrial membrane (TOM complex). Different views exist about the secondary structure and electrophysiological characteristics of Tom40 from Saccharomyces cerevisiae and Neurospora crassa. We have directly compared expressed and renatured Tom40 from both species and find a high content of beta-structure in circular dichroism measurements in agreement with refined secondary structure predictions. The electrophysiological characterization of renatured Tom40 reveals the same characteristics as the purified TOM complex or mitochondrial outer membrane vesicles, with two exceptions. The total conductance of the TOM complex and outer membrane vesicles is twofold higher than the total conductance of renatured Tom40, consistent with the presence of two TOM pores. TOM complex and outer membrane vesicles possess a strongly enhanced sensitivity to a mitochondrial presequence compared to Tom40 alone, in agreement with the presence of several presequence binding sites in the TOM complex, suggesting a role of the non-channel Tom proteins in regulating channel activity.

Binding Sites↗
PubMed1975

In vitro reassembly of the membranous vesicle from Escherichia coli outer membrane components. Role of individual components and magnesium ions in reassembly.

A method was developed for the reassembly of membranous vesicle from the sodium deoxycholate-dissociated outer membrane components of Escherichia coli. The removal of the detergent by dialysis and the presence of Mg2+ were essential for the reassembly. Membrane protein alone did not form any membranous structure. Closed membranous vesicles similar to the native outer membrane were reassembled only when protein was mixed with both lipopolysaccharide and phospholipid in deoxycholate solution and subsequently dialized. The membrane showed a distinct trilaminar structure with a center-to-center distance between two dark lines of 53 A, which is a characteristic of the native outer membrane. This characteristic trilaminar structure was shown to be due to the presence of lipopolysaccharide. Phospholipd was required for the vesicularization of membrane. Lipopolysaccharide and/or phospholipid formed a membranous structure in the absence of protein, while the morphology of their negatively stained sample was quite different from that of the native outer membrane unless the outer membrane protein was added to the reassembly mixture. The protein from the cytoplasmic membrane was unable to reform membranous vesicle with lipopolysaccharide and phospholipid, indicating that the reassembly system discriminated outer membrane proteins from cytoplasmic proteins.

Bacterial Proteins↗
PubMed2005

Cross-linking analysis of antigenic outer membrane protein complexes of Neisseria meningitidis.

Polysaccharide-based approaches have not enabled the development of effective vaccines against meningococci of serogroup B, and the most promising current research is focused on the use of outer membrane vesicles. Due to the toxicity of the outer membrane oligosaccharides, new vaccines based on purified proteins are being sought, but despite the application of advanced techniques, they remain elusive, perhaps due to the fact that standard techniques for analysis of antigens overlook conformational epitopes located in membrane complexes. Membrane complex antigens have been analyzed in Neisseria gonorrhoeae, and a study published on Neisseria meningitidis has reported the in vitro formation of 800-kD complexes by deposition of a purified protein (MSP63) onto synthetic lipid layers; however, no studies to date have attempted to identify membrane complexes present in vivo in N. meningitidis. In the present study, cross-linking with formaldehyde was used to identify outer membrane protein associations in various N. meningitidis and Neisseria lactamica strains. In N. meningitides, complexes of about 450 kD (also present in N. lactamica), 165 and 95 kD were detected and shown to be made up of the proteins MSP63, PorA/PorB/RmpM/FetA, and PorA/PorB/RmpM, respectively. In western blots, the 450-kD complex was identified by mouse antibodies raised against outer membrane vesicles, but not by antibodies raised against the purified complex, demonstrating the importance of conformational epitopes, and thus suggesting that the analysis of antigens in their native conformation may be useful or even essential for the design of effective vaccines against meningococci.

Antigens, Bacterial↗
PubMed1989

Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger.

The aim of this study was to identify and purify the Na+-H+ exchanger from rabbit renal brush border membranes by use of affinity chromatography. Triton-solubilized membranes were equilibrated with an affinity matrix consisting of the amiloride analogue A35 (5-N-(3-aminophenyl)amiloride) covalently coupled to Sepharose CL-4B beads through a triglycine spacer arm. The matrix was then washed extensively with buffer and sequentially eluted with buffer, buffer containing 5 mM amiloride, and 1% sodium dodecyl sulfate (SDS). Eluates were concentrated and subjected to SDS-polyacrylamide gel electrophoresis. The silver-stained gel revealed a 25-kDa protein that was not visible in the initial solubilized brush border membrane extract, was not eluted from the affinity matrix by buffer alone, but was eluted with 5 mM amiloride. A subsequent elution with 1% SDS did not release any more of the 25-kDa protein, indicating that it had been completely eluted from the affinity matrix by amiloride. The presence of 5 mM amiloride during equilibration of the solubilized brush border extract with the affinity matrix completely blocked adsorption of the 25-kDa protein. The relative abundance of this protein correlated closely with Na+-H+ exchange activity when preparations of cortical brush border membrane vesicles, outer medullary brush border membrane vesicles, and cortical basolateral membrane vesicles were compared. Moreover, binding of the protein to the affinity matrix was inhibited by amiloride and amiloride analogues with a rank order identical to that for inhibition of Na+-H+ exchange activity. These findings strongly suggest that the 25-kDa protein is a structural component of the Na+-H+ exchanger.

Amiloride↗
PubMed2003

Evidence for naturally acquired T cell-mediated mucosal immunity to Neisseria meningitidis.

Naturally acquired protective immunity against Neisseria meningitidis is thought to partially explain the disparity between the high levels of carriage in the human nasopharynx and the rare incidence of disease. To investigate this immunity to Neisseria meningitidis at the mucosal level, in vitro cellular responses to outer membrane vesicle preparations derived from this pathogen were examined using mononuclear cells from the palatine tonsils of adults and children. Characterization of these responses was achieved by depletion of CD45RA(+), CD45RO(+), and CD19(+) populations and outer membrane vesicles derived from isogenic mutants expressing different serosubtypes of the major outer membrane protein, porin A (PorA), no PorA and membrane preparations from a mutant with no LPS (LpxA(-)). The magnitude of cellular proliferative responses against the outer membrane vesicles were strongly associated with age and were largely T cell mediated, involving both CD45RO(+) and CD45RA(+) T cell phenotypes. Responses were not dependent on LPS but consisted of both PorA cross-specific and non-PorA-dependent responses. Cellular immunity against Neisseria meningitidis was found to be frequently associated with systemic IgG Abs but was not associated with serum bactericidal Abs. For the first time our results demonstrate an age-associated acquisition of mucosal T effector/memory cell responses to Neisseria meningitidis. This mucosal cellular immunity can be present in the absence of serum bactericidal Abs, a classical marker of protective immunity.

Adolescent↗
PubMed1993

Meningococcal lipopolysaccharide (LPS)-derived oligosaccharide-protein conjugates evoke outer membrane protein- but not LPS-specific bactericidal antibodies in mice: influence of adjuvants.

Meningococcal lipopolysaccharide (LPS)-derived oligosaccharides (OS) were coupled to tetanus toxoid (TT) and purified P1.7,16 outer membrane proteins (OMP). The immunogenicities of the conjugates with and without the addition of the adjuvant Quil A or the nonionic block polymer L121 were studied in mice. Immunotype L2 and L3,7,9 OS-TT conjugates induced immunoglobulin G (IgG) responses that were strongly augmented by Quil A and L121. These adjuvants not only enhanced the amount of IgG evoked but also shifted the IgG subclass distribution from mainly IgG1 toward the complement-activating subclasses IgG2a and IgG2b. The antibodies induced were directed against the OS part of meningococcal LPS. They were not bactericidal for group B meningococci. Both the L3,7,9 OS-P1.7,16 OMP conjugate and purified P1.7,16 OMP evoked a strong IgG response against the P1.7,16 OMP but not against the L3,7,9 LPS. These anti-OMP IgG responses were comparable to the IgG OMP-specific responses induced by the H44/76 or HIII-5 outer membrane vesicles but still did not lyse group B meningococcal strains. The IgG response evoked with OS-OMP or purified OMP consisted mainly of the IgG1 subclass, whereas the H44/76 or HIII-5 outer membrane vesicles induced high amounts of bactericidal IgG2a and IgG2b antibodies next to the IgG1 antibodies. The addition of the adjuvant Quil A or L121 to OS-OMP or OMP resulted in the induction of high levels of bactericidal anti-P1.7,16-specific OMP antibodies, as reflected by the presence of substantial amounts of IgG2a and IgG2b antibodies. These results indicate that (i) mouse anti-LPS antibodies evoked by LPS-derived OS-protein conjugates are not bactericidal for group B meningococci, (ii) extensive purification of P1.7,16 OMP can lead to the loss of the intrinsic adjuvant properties of outer membrane vesicle preparations, and (iii) the addition of suitable adjuvants restores the ability of these purified P1.7,16 OMP to induce bactericidal antibodies.

Adjuvants, Immunologic↗
PubMed1992

Porphyromonas gingivalis virulence in mice: induction of immunity to bacterial components.

Selected cell envelope components of Porphyromonas gingivalis were tested in a BALB/c mouse model in an attempt to elucidate further the outer membrane components of this putative oral pathogen that might be considered as virulence factors in host tissue destruction. Lipopolysaccharide (LPS), outer membrane, and outer membrane vesicles of P. gingivalis W50, ATCC 53977, and ATCC 33277 were selected to examine an immunological approach for interference with progressing tissue destruction. Mice were actively immunized with heat-killed (H-K) or Formalin-killed (F-K) whole cells or with the outer membrane fraction, LPS, or outer membrane vesicles of the invasive strain P. gingivalis W50. The induction of invasive spreading lesions with tissue destruction and lethality were compared among different immunization groups in normal, dexamethasone-treated (dexamethasone alters neutrophil function at the inflammatory site), and galactosamine-sensitized (galactosamine sensitization increases endotoxin sensitivity) mice after challenge infection with the homologous strain (W50) and heterologous strains (ATCC 53977 and ATCC 33277). Enzyme-linked immunosorbent assay analyses revealed significantly elevated immunoglobulin G and M antibody responses after immunization with H-K or F-K cells or the outer membrane fraction compared with those of nonimmunized mice. The killed whole-cell vaccines provided significantly greater protection against challenge infection in normal mice (decreased lesion size and death) than did either the outer membrane fraction or LPS immunization. The lesion development observed in dexamethasone-pretreated mice was significantly enhanced compared with that of normal mice after challenge with P. gingivalis. Immunization with P. gingivalis W50 provided less protection against heterologous challenge infection with P. gingivalis ATCC 53977; however, some species-specific antigens were recognized and induced protective immunity. Only viable P. gingivalis induced a spreading lesion in normal, dexamethasone-treated, or galactosamine-sensitized mice; F-K or H-K bacteria did not induce lesions. The F-K and outer membrane vesicle immunization offered greater protection from lesion induction than did the H-K immunogen after challenge infection simultaneous with galactosamine sensitization. The H-K cell challenge with galactosamine sensitization produced 100% mortality without lesion induction, suggesting that LPS or LPS-associated outer membrane molecules were functioning like endotoxin. Likewise, P. gingivalis W50 LPS (1 micrograms per animal) administered intravenously produced 80% mortality in galactosamine-sensitized mice. In contrast to the effects of immunization on lesion development, immunization with H-K or F-K cells or LPS provided no protection against intravenous challenge with LPS; 100% of the mice died from acute endotoxin toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1981

Isolation and partial characterization of outer and inner membranes from encapsulated Haemophilus influenzae type b.

A method has been developed to separate the cell envelope of encapsulated (type b) Haemophilus influenzae into its outer and inner membrane components with procedures that avoided two problems encountered in fractionation of this envelope: (i) the tendency of the outer and inner membranes to hybridize and (ii) the tendency of the apparently fragile inner membrane to fragment into difficulty sedimentable units. Log phage cells, whose lipids were radioactively labeled, were lysed by passage through a French press. The lysate was applied to a discontinuous sucrose gradient, and envelope-rich material was collected by centrifugation onto a cushion of dense sucrose under carefully controlled conditions. This material was then further fractionated by isopycnic centrifugation in a sucrose gradient to yield four membrane fractions which were partially characterized. On the basis of their radioactivity, buoyant density, ultrastructure, polypeptide composition, and content of phospholipid, protein, lipopolysaccharide, and succinic dehydrogenase, these fractions were identified as follows: fraction 1, outer membrane vesicles with very little inner membrane contamination (less than 4%); fraction 2, outer membrane vesicles containing entrapped inner membrane; fraction 3, a protein-rich fraction of inner membrane; fraction 4, a protein-poor fraction of inner membrane. Fractions 3 and 4 contained about 25% outer membrane contamination.

Bacterial Proteins↗
PubMed1990

Characterization of FadL-specific fatty acid binding in Escherichia coli.

The product of the fadL gene (FadL) is a central component of the long-chain fatty acid transport system of Escherichia coli. When fatty acid activation is blocked by a mutation in the structural gene for acyl CoA synthetase (fadD) transport is inhibited allowing a FadL-specific fatty acid binding activity to be measured. This binding activity was 4- to 6-fold greater in the fadL+ fadD strain LS6928 when compared to the delta fadLfadD strain LS6929. With long-chain fatty acids, this binding activity was saturable and it was estimated that there were approx. 35,000 FadL-specific oleic acid binding sites per cell in the fadL+ strain LS6928. The FadL-specific fatty acid binding affinity was highest for oleic acid (18:1) and palmitic acid (16:0) giving apparent KD values of 2.3.10(-7) M and 8.8.10(-7) M, respectively. FadL-specific binding affinity of myristic acid (14:0) was nearly an order of magnitude less and no FadL-specific binding of decanoic acid (10:0) could be measured. Two lines of evidence suggest that FadL-fatty acid binding occurs by a hydrophobic interaction: (1) There was a preference for the long-chain substrates oleic acid and palmitic acid; and (2) oleic acid binding activity was not significantly changed over the pH range 5.0 to 8.0. The FadL-specific binding of oleic acid in the fadL+ strain LS6928 could be blocked by preincubation with antisera raised against purified FadL providing a clear correlation between the activity and identity of FadL. The binding activity associated with FadL was measured in vesicles of the outer membrane following passage over the hydrophobic resin Lipidex 1000. The KD of oleic acid binding attributable to FadL in outer membranes vesicles (6.0.10(-7) M) was in close agreement with that determined in whole cells. Overall, these studies demonstrated that FadL binds long-chain fatty acids with a relatively high affinity prior to their transport across the outer membrane.

Bacterial Outer Membrane Proteins↗
PubMed1993

Humoral immune response to the class 3 outer membrane protein during the course of meningococcal disease.

We have determined the amounts of specific anti-class 3 outer membrane protein antibodies of immunoglobulin G (IgG), IgM, and IgA isotypes in patient sera during the course of meningococcal disease by using purified class 3 protein as the sensitizing antigen in an enzyme-linked immunosorbent assay. The class 3 protein was obtained from a variant of strain 44/76 (B:15:P1.7,16) lacking class 1 and class 4 outer membrane proteins. Serum samples from 25 patients with systemic meningococcal disease caused by organisms of various serotypes were collected during the course of disease. Seven of these patients had been immunized with a meningococcal outer membrane vesicle vaccine made from strain 44/76 prior to disease. An increase in specific anti-class 3 (type 15) outer membrane protein IgG antibodies was demonstrated in 22 of 25 patients (88%), regardless of the serotype of the infecting strain. This indicates that the specific anti-class 3 antibodies were reacting in part with epitopes not determined by the monoclonal antibodies used for serotyping. A considerable heterogeneity in antibody levels and IgG subclass response was seen. Most patients had low levels of anti-class 3 antibodies during the acute illness, with antibodies peaking during the second week of disease and returning to near baseline in sera collected 6 to 12 months after the onset of the disease. The majority of the specific anti-class 3 IgG antibodies were shown to bind to surface-exposed epitopes on the whole bacteria and to belong to IgG1 and IgG3. The highest anti-class 3 IgG peak levels were seen in patients infected with strains of the homologous serotype after vaccination with the meningococcal outer membrane vesicle vaccine, suggesting an anamnestic response. However, these patients were not protected from meningococcal disease after immunization.

Adolescent↗
PubMed1986

Molecular cloning of DNA coding for outer membrane proteins of Haemophilus influenzae type b.

DNA from Haemophilus influenzae type b was cloned in Escherichia coli with a vector lambda gt11 Amp1. Clones producing antigens reactive with hyperimmune rabbit antisera were identified by colony radioimmune assay. A second screening with hyperimmune serum adsorbed with intact H. influenzae type b bacteria was used to identify those clones producing surface-exposed outer membrane proteins. The proteins expressed in E. coli were coupled to Sepharose and used to affinity purify antibodies which were tested for reactivity with outer membrane vesicles. It was found by Western blotting that the clones were producing antigens corresponding to Mr 49,000, 39,500, and 35,000 major outer membrane protein or antigens of H. influenzae type b. Additional clones could be detected by human serum, but their reactivity was not removed when serum was adsorbed with intact bacteria. One of these studied in more detail was found to produce an antigen present in H. influenzae type b lysate but not in outer membrane vesicle preparations.

Antigens, Bacterial↗
PubMed2000

A role for the bacterial outer membrane in the pathogenesis of Helicobacter pylori infection.

Helicobacter pylori infection in humans is associated with diverse of clinical outcomes which are partly attributed to bacterial strain differences. Secreted bacterial products are thought to be involved in the pathogenesis caused by this non-invasive bacterium. Electron microscopy of gastric biopsies from infected individuals revealed blebbing of the H. pylori outer membrane, similar to the process of outer membrane vesicle shedding which occurs when the bacterium is grown in broth. Porins, a class of proinflammatory proteins, were observed in the outer membrane vesicles. The VacA cytotoxin, which is produced by 50-60% of H. pylori strains and associated with increased pathogenesis of infection, was also found to be vesicle-associated and biologically active. This supports the hypothesis that these vesicles represent a vehicle for the delivery of damaging bacterial products to the gastric mucosa.

Bacterial Outer Membrane Proteins↗
PubMed1993

Effect of immunization of pulmonary clearance of Moraxella catarrhalis in an animal model.

A murine model for pulmonary clearance of Moraxella catarrhalis was used to determine whether immunization could enhance clearance of this organism from the lungs. Animals actively immunized with outer membrane vesicles of M. catarrhalis cleared an endobronchial challenge with the homologous strain more quickly than did sham-immunized control animals. Western blot analysis of both this immune mouse serum and rabbit antiserum raised against outer membrane vesicles of M. catarrhalis indicated that antibodies were present to both outer membrane protein and lipooligosaccharide antigens. Passive immunization of mice with the immune rabbit serum resulted in enhanced pulmonary clearance of both homologous and heterologous strains of M. catarrhalis, indicating the involvement of serum antibody in this clearance process and the existence of conserved surface antigens in the two different M. catarrhalis strains. These results suggest that this model system may be useful for the identification of vaccine candidates among the surface antigens of M. catarrhalis.

Animals↗
PubMed1993

Phospholipid asymmetry of the outer membrane of rat liver mitochondria. Evidence for the presence of cardiolipin on the outside of the outer membrane.

The phospholipid topology of the outer membrane of intact rat liver mitochondria and derived outer membrane vesicles was investigated by determining the accessible pool of the various phospholipid classes towards phospholipase A2, a phosphatidylcholine-specific transfer protein and by chemical labeling using trinitrobenzene sulfonic acid. The outer membrane vesicles are sealed and have a right-side-out topology with a proposed localization of 55%, 77%, 100%, and at least 30% of the phosphatidylcholine, phosphatidylethanolamine, cardiolipin, and phosphatidylinositol plus phosphatidylserine in the outer leaflet, respectively. The outer membrane in intact mitochondria appears to have a similar phospholipid distribution.

Animals↗
PubMed2001

Identification of phospholipids as new components that assist in the in vitro trimerization of a bacterial pore protein.

The in vitro trimerization of folded monomers of the bacterial pore protein PhoE, into its native-like, heat- and SDS-stable form requires incubations with isolated cell envelopes and Triton X-100. The possibility that membranes could be isolated that are enriched in assembly factors required for assembly of the pore protein was now investigated. Fractionation of total cell envelopes of Escherichia coli via various techniques indeed revealed the existence of membrane fractions with different capacities to support assembly in vitro. Fractions containing mainly inner membrane vesicles supported the formation of trimers that were associated with these membrane vesicles. However, only a proportion of these trimers were heat- and SDS-stable and these were formed with slow kinetics. In contrast, fractions containing mainly outer membrane vesicles supported formation of high amounts of heat-stable trimers with fast kinetics. We identified phospholipids as active assembly components in these membranes that support trimerization of folded monomers in a process with similar characteristics as observed with inner membrane vesicles. Furthermore, phospholipids strongly stimulate the kinetics of trimerization and increase the final yield of heat-stable trimers in the context of outer membranes. We propose that lipopolysaccharides stabilize the assembly competent state of folded monomers as a lipochaperone. Phospholipids are involved in converting the folded monomer into new assembly competent intermediate with a short half-life that will form heat-stable trimers most efficiently in the context of outer membrane vesicles. These results provide biochemical evidence for the involvement of different lipidic components at distinct stages of the porin assembly process.

Centrifugation, Density Gradient↗
PubMed2000

Cellular activating properties and morphology of membrane-bound and purified meningococcal lipopolysaccharide.

Neisseria meningitidis, the cause of epidemic meningitis and acute lethal sepsis, synthesizes surplus lipopolysaccharides (LPSs) during growth, which are released as outer membrane vesicles (OMV) or "blebs". Meningococcal disease severity is related to plasma LPS levels. We have compared the biological activities of native outer membrane vesicles (nOMV) to those of purified Nm-LPS (Nm-LPS) and LPS-depleted OMV (dOMV) prepared from N. meningitidis. The LPS content of nOMV was determined spectrophotometrically by quantifying KDO and by silver-stained SDS-PAGE gels. The morphology of the preparations was studied by transmission electron microscopy. The Limulus amoebocyte lysate (LAL) assay was used to quantify LPS in the plasma solutions. The preparations were diluted in endotoxin-free heparin plasma to equal amounts of LPS (w/w) in the range 50-5000 pg/ml. The biological reactivity was tested by: (i) a monocyte target-assay (monocyte purity > or =96%); and (ii) a whole blood model, measuring the secretion of TNF-alpha and IL-6 induction of procoagulant activity in monocytes (PCA). In both models, nOMV induced dose-dependent cell responses (TNF-alpha, IL-6, PCA) similar to purified Nm-LPS, whereas dOMV induced minimal responses. However, LAL activity was significantly higher for nOMV than for purified Nm-LPS and dOMV. The cellular responses of purified Nm-LPS and nOMV were reduced (>95%) by a specific anti-CD14-antibody.

Adult↗
PubMed1987

Malonyl-CoA binding site and the overt carnitine palmitoyltransferase activity reside on the opposite sides of the outer mitochondrial membrane.

The overt carnitine palmitoyltransferase (palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21) activity of intact mitochondria from rat heart and liver was found to be resistant to the action of proteases such as Nagarse (subtilisin, EC 3.4.21.14). Nagarse under the same conditions, however, greatly decreased the malonyl-CoA inhibition of carnitine palmitoyltransferase activity, the high-affinity binding of malonyl-CoA to mitochondria, and the ability of malonyl-CoA to shift to the right the sigmoid activity curve of carnitine palmitoyltransferase observed with variations in palmitoyl-CoA concentration. No noticeable effect of Nagarse pretreatment was observed on the binding of octanoyl-CoA to mitochondria. Subfractionation of liver mitochondria using a combination of swelling, shrinking, and density gradient centrifugation yielded a membrane fraction in which the specific activities of the outer membrane marker enzymes were enriched greater than or equal to 16-fold together with a near-parallel enrichment of malonyl-CoA-inhibitable carnitine palmitoyltransferase activity. The percent recovery of this carnitine palmitoyltransferase in the outer membrane vesicles also matched that of the known outer membrane markers. The carnitine palmitoyltransferase activity of these out-side-out vesicles became susceptible to added Nagarse only on their cosonication. These findings show that whereas the malonyl-CoA binding site relevant to the inhibition of carnitine palmitoyltransferase is situated on the outer side of the outer membrane, the overt carnitine palmitoyltransferase activity resides on the inner side of the outer membrane.

Acyl Coenzyme A↗
PubMed2000

Characteristics of Helicobacter pylori attachment to human primary antral epithelial cells.

Conventional cell lines are commonly used to study infection characteristics of the human gastric pathogen Helicobacter pylori. We sought to investigate bacterial attachment to human antral primary epithelial cells, a cell model that more closely resembles the human stomach than transformed cell lines. Primary cells were infected for 24 and 48 h with H. pylori. Morphological appearance of both the pathogen and the cells as well as features of colonization, attachment and internalization were evaluated by electron microscopy and compared to features observed with cultured AGS cells. H. pylori exhibited various shapes during colonization including the spiral, U-shaped, donut, and coccoid forms. The prevalence of each form seemed to be dependent on the infected donor tissue but, in general, changed with time to the coccoid form. Bacterial cell membranes progressively enlarged and appeared at times to be connected with microvilli. Bacterial attachment occurred to cells that were either unchanged, or had formed cup-like structures. Simultaneously, outer membrane vesicles were increasingly secreted from the bacteria, coinciding with increased cellular damage. We conclude that bacterial shape conversion, adherence and secretion of outer membrane vesicles are features of H. pylori infection. Primary gastric cell cultures closely imitate the antral environment and present an appropriate and useful model to study H. pylori pathogenesis.

Bacterial Adhesion↗