Molecular and functional characterisation of the Serratia marcescens outer membrane protein Omp1

Serratia marcescens outer membrane contains three different general diffusion porins: Omp1, Omp2 and Omp3. Omp1 was cloned and sequenced and it shows a great homology to the family of outer membrane porins that comprises the general porins of enteric bacteria. The gene for Omp1 was transferred into an expression plasmid and was expressed in Escherichia coli UH302 (E. coli UH302 pOM100), a porin deficient strain. Its expression confers a higher susceptibility towards different antibiotics to this strain. Omp1 was purified to homogeneity from outer membrane of E. coli UH302 pOM100. Reconstitution of the purified protein into black lipid bilayers demonstrated that it is a channel-forming component with a single-channel conductance of approximately 2 nS in 1 M KCl similar to that of other porins from enteric bacteria. Omp1 is slightly cation-selective. Its homology to already crystallised members of the family of enteric porins whose three-dimensional-structures are known and allowed the design of a topology model for Omp1. The charge distribution within a porin monomer is similar as in other general diffusion pores. The positively charged amino acids localised at the beta-strands opposite the external loop L3, which restrict the pore diameter in the porin monomer.     

Acinetobacter baumannii outer membrane protein 34 elicits NLRP3 inflammasome activation via mitochondria-derived reactive oxygen species in RAW264.7 macrophages

Acinetobacter baumannii (A. baumannii) is a Gram-negative bacterium, which acts as an opportunistic pathogen and causes hospital-acquired pneumonia and bacteremia by infecting the alveoli of epithelial cells and macrophages. Evidence reveals that A. baumannii outer membrane protein 34 (Omp34) elicits cellular immune responses and inflammation. The innate immunity NOD-like receptor 3 (NLRP3) inflammasome exerts critical function against pneumonia caused by A. baumannii infection, however, the role of Omp34 in the activation of the NLRP3 inflammasome and its corresponding regulatory mechanism are not clearly elucidated. The present study aimed to investigate whether Omp34 elicited NLRP3 inflammasome activation through the mitochondria-derived reactive oxygen species (ROS). Our results showed that Omp34 triggered cell pyroptosis by up-regulating the expression of NLRP3 inflammasome-associated proteins and IL-1β release in a time- and dose-dependent manner. Omp34 induced the expression of caspase-1-p10 and IL-1β, which was significantly attenuated by NLRP3 gene silencing in RAW264.7 mouse macrophage cells. Additionally, Omp34 stimulated RAW264.7 mitochondria to generate ROS, while the ROS scavenger Mito-TEMPO inhibited the Omp34-triggered expression of NLRP3 inflammasome-associated proteins and IL-1β synthesis. The above findings indicate that mitochondria-derived ROS play an important role in the process of NLRP3 inflammasome activation. In summary, our study demonstrates that the A. baumannii pathogen pattern recognition receptor Omp34 activates NLRP3 inflammasome via mitochondria-derived ROS in RAW264.7 cells. Accordingly, down-regulating the mitochondria-derived ROS prevents the severe infection consequences caused by A. baumannii-induced NLRP3 inflammasome hyper-activation.     

Eicosapentaenoic acid facilitates the folding of an outer membrane protein of the psychrotrophic bacterium, Shewanella livingstonensis Ac10

Polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), are found in various cold-adapted microorganisms. We previously demonstrated that EPA-containing phospholipids (EPA-PLs) synthesized by the psychrotrophic bacterium Shewanella livingstonensis Ac10 support cell division, membrane biogenesis, and the production of membrane proteins at low temperatures. In this article, we demonstrate the effects of EPA-PLs on the folding and conformational transition of Omp74, a major outer membrane cold-inducible protein in this bacterium. Omp74 from an EPA-less mutant migrated differently from that of the parent strain on SDS-polyacrylamide gel, suggesting that EPA-PLs affect the conformation of Omp74 in vivo. To examine the effects of EPA-PLs on Omp74 protein folding, in vitro refolding of recombinant Omp74 was carried out with liposomes composed of 1,2-dipalmitoleoyl-sn-glycero-3-phosphoglycerol and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (1:1molar ratio) with or without EPA-PLs as guest lipids. SDS-PAGE analysis of liposome-reconstituted Omp74 revealed more rapid folding in the presence of EPA-PLs. CD spectroscopy of Omp74 folding kinetics at 4°C showed that EPA-PLs accelerated β-sheet formation. These results suggest that EPA-PLs act as chemical chaperones, accelerating membrane insertion and secondary structure formation of Omp74 at low temperatures.     

The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane

In Gram-negative bacteria, lipopolysaccharide and phospholipid biosynthesis takes place at the inner membrane. How the completed lipid molecules are subsequently transported to the outer membrane remains unknown. Omp85 of Neisseria meningitidis is representative for a family of outer membrane proteins conserved among Gram-negative bacteria. We first demonstrated that the omp85 gene is co-transcribed with genes involved in lipid biosynthesis, suggesting an involvement in lipid assembly. A meningococcal strain was constructed in which Omp85 expression could be switched on or off through a tac promoter-controlled omp85 gene. We demonstrated that the presence of Omp85 is essential for viability. Depletion of Omp85 leads to accumulation of electron-dense amorphous material and vesicular structures in the periplasm. We demonstrated, by fractionation of inner and outer membranes, that lipopolysaccharide and phospholipids mostly disappeared from the outer membrane and instead accumulated in the inner membrane, upon depletion of Omp85. Omp85 depletion did not affect localization of integral outer membrane proteins PorA and Opa. These results provide compelling evidence for a role for Omp85 in lipid transport to the outer membrane.     

Expression, two-dimensional crystallization, and three-dimensional reconstruction of the beta8 outer membrane protein Omp21 from Comamonas acidovorans

The Omp21 protein from the proteobacterium Comamonas (Delftia) acidovorans belongs to the recently described beta8 family of outer membrane proteins, characterized by eight antiparallel beta-strands which form a beta-barrel. This family includes virulence proteins, OmpA and OmpX from Escherichia coli, and other related molecules. After we established an expression system, recombinant Omp21 was purified by Ni(2+) chelation affinity chromatography and refolded in situ while bound to resin. The native state of refolded protein was proven by FTIR spectroscopy and monitored with denaturing PAGE (heat modification). Both native and recombinant Omp21 were reconstituted in lipid membranes and crystallized two-dimensionally by controlled dialysis. Recombinant Omp21 crystallized as dimer and formed a p22(1)2(1) lattice with constants of a = 11.1 nm, b = 12.2 nm, gamma = 89.5 degrees. The 3-D structure of negatively stained, recombinant Omp21 was determined at a resolution of 1.8 nm by means of electron crystallography. Comparison with 3-D maps of OmpX and the transmembrane domain of OmpA revealed a high similarity between the mass distribution of exoplasmic loops of Omp21 and OmpA.     

Porin Involvement in Cephalosporin and Carbapenem Resistance of Burkholderia pseudomallei

Background

Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins.

Principal Findings

Microbiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the ‘greasiness’ of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive.

Conclusion/Significance

Our results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents.     

Molecular, antigenic, and functional analyses of Omp2b porin size variants of Brucella spp

Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various Brucella species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of Brucella melitensis reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus Brucella seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of Brucella suis biovar 3), the porin variability does not result in major antigenic variability of the Brucella surface that could help the bacteria during the reinfection of a host. Porin variation in Brucella seems to result mainly in porin conductivity modifications.     

Localization of a Porphyromonas gingivalis 26-kilodalton heat-modifiable, hemin-regulated surface protein which translocates across the outer membrane.

We recently identified a 26-kDa hemin-repressible outer membrane protein (Omp26) expressed by the periodontal pathogen Porphyromonas gingivalis. We report the localization of Omp26, which may function as a component of a hemin transport system in P. gingivalis. Under hemin-deprived conditions, P. gingivalis expressed Omp26, which was then lost from the surface after a shift back into hemin-rich conditions. Experiments with 125I labeling of surface proteins to examine the kinetics of mobilization of Omp26 determined that it was rapidly (within less than 1 min) lost from the cell surface after transfer into a hemin-excess environment. When cells grown under conditions of hemin excess were treated with the iron chelator 2,2'-bipyridyl, Omp26 was detected on the cell surface after 60 min. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses using purified anti-Omp26 monospecific polyclonal immunoglobulin G antisera established that Omp26 was heat modifiable (39 kDa unheated) and consisted of a single protein species. Immunogold labeling of negatively stained and chemically fixed thin-section specimens indicated that Omp26 was associated with the cell surface and outer leaflet of the P. gingivalis outer membrane in hemin-deprived conditions but was buried in the deeper recesses of the outer membrane in hemin-excess conditions. Analysis of subcellular fractions of P. gingivalis grown either in hemin-excess or hemin-deprived conditions detected Omp26 only in the cell envelope fraction, not in the cytoplasmic fraction or culture supernatant. Limited proteolytic digestion of hemin-deprived P. gingivalis with trypsin and proteinase K verified the surface location of Omp26 as well as its susceptibility to proteolytic digestion. Heat shock treatment of hemin-excess-grown P. gingivalis also resulted in Omp26 translocation onto the outer membrane surface even in the presence of hemin. Furthermore, hemin repletion of heat-shocked, hemin-deprived P. gingivalis did not result in Omp26 translocation off the outer membrane surface, suggesting that thermal stress inactivates this transmembrane event. This newly described outer membrane protein appears to be associated primarily with the outer membrane, in which it is exported to the outer membrane surface for hemin binding and may be imported across the outer membrane for intracellular hemin transport.     

Preliminary studies of the 2D crystallization of Omp1 of Serratia marcescens: observation by atomic force microscopy in native membranes environment and reconstituted in proteolipid sheets

In this work the porin Omp1 of Serratia marcescens was expressed in a porin deficient mutant (Escherichia coli UH302) and its functionality studied following the accumulation of ciprofloxacin in bacteria. The protein was extracted, purified and reconstituted in proteoliposomes of different composition (lipopolysaccharide (LPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)). Maximum extraction of the detergent was achieved applying different steps of dialysis and centrifugation. Proteolipid sheets with different composition were spread onto mica and observed by atomic force microscopy. Two-dimensional crystal of Omp1 was not observed in any case due to low resolution achieved. Judging from the images features POPC is the most suitable phospholipid to enhance 2D lattice formation for Omp1.     

Importance of the Omp25/Omp31 family in the internalization and intracellular replication of virulent B. ovis in murine macrophages and HeLa cells

The role of the outer membrane proteins of the Omp25/Omp31 family in invasiveness and intracellular survival of virulent B. ovis in phagocytes was analyzed. The absence of Omp25d or Omp22 in B. ovis abolished its invasive capacity in HeLa cells and reduced it in J774.A1 cells. Additionally, in J774.A1 cells, the Deltaomp25d mutant was unable to multiply, whereas the Deltaomp22 mutant was cleared at 24h post-infection. These findings demonstrate that Omp25d and Omp22 are essential for the invasion and survival of B. ovis inside host cells, and justify the strong attenuation in virulence of the Deltaomp25d and Deltaomp22 mutants.     

Identification of a novel heterodimeric outer membrane protein of Porphyromonas gingivalis by two-dimensional gel electrophoresis and peptide mass fingerprinting.

Porphyromonas gingivalis is a Gram-negative, anaerobic bacterium associated with chronic periodontitis. A 2D electrophoretic analysis of the outer membrane of P. gingivalis W50 revealed a dominant train of spots at 40-41 kDa. The proteins in the train of spots were digested in-gel with trypsin and identified by MS. The train of spots represented two proteins, designated Omp40 and Omp41 that share 47% sequence identity. Preparation of outer membranes in the absence of protease inhibitors resulted in partial cleavage of Omp40 and Omp41 to produce an N-terminal and C-terminal fragment of both proteins. The N-terminal fragments displayed the same isoelectric heterogeneity as the intact proteins. Almost 100% of the amino-acid sequence of these N-terminal fragments in each 2D gel spot was verified suggesting lack of post-translational modification. Re-subjecting a single N-terminal domain spot to 2D electrophoresis resulted in the complete series of spots being reproduced, suggesting that the heterogeneity was related to conformational equilibria. Under reduced conditions and without heating, Omp40 and Omp41 migrated as 34- to 35-kDa proteins in SDS/PAGE whereas under nonreduced conditions the proteins migrated as 70-kDa proteins, suggesting the formation of dimers through intersubunit disulfide bonds. The proteins each contain two cysteine residues in the conserved sequence RPVSCPECPE. Tryptic peptides generated from the nonreduced forms of the proteins confirmed the presence of heterodimers stabilized through intersubunit disulfide bond formation. With the exception of heterodimer formation, the two proteins share several similarities with OmpA-like porins of other Gram-negative bacteria including consensus sequence, abundance, modification by heat, overall length and positioning of domains.     

Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies

Outer membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS), the main surface antigens of Brucella ovis, display surface-exposed epitopes. Mixtures of monoclonal antibodies (mAbs) to both antigens were previously shown to protect mice against a B. ovis challenge. To further identify the antigens involved, seven mAbs against Brucella OMPs (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b and Omp1) and three to R-LPS were tested for protection either individually or in combinations. Significant reduction in spleen infection in challenged mice, relative to controls, was used as the protection criteri. Controls included nonimmunized mice and mice given an irrelevant, anti-O-polysaccharide (OPS), mAb. For comparison, a group received a mouse serum containing antibodies to both OMPs and R-LPS; this serum was prepared by immunization with a B. ovis hot-saline extract which, as described previously, induces protective immunity in mice and rams. Significant protection was observed with both mAbs to OMPs and R-LPS. mAbs to Omp16, Omp19 and Omp31 afforded the highest protection and prevented the development of splenomegaly. The protective effect of mAb to Omp31 was not interfered with by nonprotective mAbs in different mixtures. The data presented confirm the protective role of antibodies to OMPs and R-LPS against B. ovis, and identify several OMPs, especially Omp31, which are promising candidates for a subunit vaccine against ram epididymitis.     

Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial infection. Despite considerable clinical and epidemiological data regarding the role of A. baumannii in nosocomial infection, the specific virulence factor or pathogenic mechanism of this organism has yet to be elucidated. This study investigated the molecular mechanism of apoptosis on the infection of human laryngeal epithelial HEp-2 cells with A. baumannii and examined the contribution of outer membrane protein 38 (Omp38) on the ability of A. baumannii to induce apoptosis of epithelial cells. A. baumannii induced apoptosis of HEp-2 cells through cell surface death receptors and mitochondrial disintegration. The Omp38-deficient mutant was not as able to induce apoptosis as the wild-type A. baumannii strain. Purified Omp38 entered the cells and was localized to the mitochondria, which led to a release of proapoptotic molecules such as cytochrome c and apoptosis-inducing factor (AIF). The activation of caspase-3, which is activated by caspase-9, degraded DNA approximately 180 bp in size, which resulted in the appearance of a characteristic DNA ladder. AIF degraded chromosomal DNA approximately 50 kb in size, which resulted in large-scale DNA fragmentation. These results demonstrate that Omp38 may act as a potential virulence factor to induce apoptosis of epithelial cells in the early stage of A. baumannii infection.     

How β-Lactam Antibiotics Enter Bacteria: A Dialogue with the Porins

Background

Multi-drug resistant (MDR) infections have become a major concern in hospitals worldwide. This study investigates membrane translocation, which is the first step required for drug action on internal bacterial targets. β-lactams, a major antibiotic class, use porins to pass through the outer membrane barrier of Gram-negative bacteria. Clinical reports have linked the MDR phenotype to altered membrane permeability including porin modification and efflux pump expression.

Methodology/Principal Findings

Here influx of β-lactams through the major Enterobacter aerogenes porin Omp36 is characterized. Conductance measurements through a single Omp36 trimer reconstituted into a planar lipid bilayer allowed us to count the passage of single β-lactam molecules. Statistical analysis of each transport event yielded the kinetic parameters of antibiotic travel through Omp36 and distinguishable translocation properties of β-lactams were quantified for ertapenem and cefepime. Expression of Omp36 in an otherwise porin-null bacterial strain is shown to confer increases in the killing rate of these antibiotics and in the corresponding bacterial susceptibility.

Conclusions/Significance

We propose the idea of a molecular “passport” that allows rapid transport of substrates through porins. Deciphering antibiotic translocation provides new insights for the design of novel drugs that may be highly effective at passing through the porin constriction zone. Such data may hold the key for the next generation of antibiotics capable of rapid intracellular accumulation to circumvent the further development MDR infections.     

YaeT (Omp85) affects the assembly of lipid-dependent and lipid-independent outer membrane proteins of Escherichia coli

The Omp85 family of proteins has been found in all Gram-negative bacteria and even several eukaryotic organisms. The previously uncharacterized Escherichia coli member of this family is YaeT. The results of this study, consistent with previous Omp85 studies, show that the yaeT gene encodes for an essential cellular function. Direct examinations of the outer membrane fraction and protein assembly revealed that cells depleted for YaeT are severely defective in the biogenesis of outer membrane proteins (OMPs). Interestingly, assemblies of the two distinct groups of OMPs that follow either SurA- and lipopolysaccharide-dependent (OmpF/C) or -independent (TolC) folding pathways were affected, suggesting that YaeT may act as a general OMP assembly factor. Depletion of cells for YaeT led to the accumulation of OMPs in the fraction enriched for periplasm, thus indicating that YaeT facilitates the insertion of soluble assembly intermediates from the periplasm to the outer membrane. Our data suggest that YaeT's role in the assembly of OMPs is not mediated through a role in lipid biogenesis, as debated for Omp85 in Neisseria, thus advocating a conserved OMP assembly function of Omp85 homologues.     

Identification of the essential Brucella melitensis porin Omp2b as a suppressor of Bax-induced cell death in yeast in a genome-wide screening

Background

Inhibition of apoptosis is one of the mechanisms selected by numerous intracellular pathogenic bacteria to control their host cell. Brucellae, which are the causative agent of a worldwide zoonosis, prevent apoptosis of infected cells, probably to support survival of their replication niche.

Methodology/Principal Findings

In order to identify Brucella melitensis anti-apoptotic effector candidates, we performed a genome-wide functional screening in yeast. The B. melitensis ORFeome was screened to identify inhibitors of Bax-induced cell death in S. cerevisiae. B. melitensis porin Omp2b, here shown to be essential, prevents Bax lethal effect in yeast, unlike its close paralog Omp2a. Our results based on Omp2b size variants characterization suggest that signal peptide processing is required for Omp2b effect in yeast.

Conclusion/Significance

We report here the first application to a bacterial genome-wide library of coding sequences of this “yeast-rescue” screening strategy, previously used to highlight several new apoptosis regulators. Our work provides B. melitensis proteins that are candidates for an anti-apoptotic function, and can be tested in mammalian cells in the future. Hypotheses on possible molecular mechanisms of Bax inhibition by the B. melitensis porin Omp2b are discussed.     

Molecular Cloning, Sequencing, and Expression of omp-40, the Gene Coding for the Major Outer Membrane Protein from the Acidophilic Bacterium Thiobacillus ferrooxidans

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.     

Molecular architecture and function of the Omp85 family of proteins

Omp85 is a protein found in Gram-negative bacteria where it serves to integrate proteins into the bacterial outer membrane. Members of the Omp85 family of proteins are defined by the presence of two domains: an N-terminal, periplasmic domain rich in POTRA repeats and a C-terminal beta-barrel domain embedded in the outer membrane. The widespread distribution of Omp85 family members together with their fundamental role in outer membrane assembly suggests the ancestral Omp85 arose early in the evolution of prokaryotic cells. Mitochondria, derived from an ancestral bacterial endosymbiont, also use a member of the Omp85 family to assemble proteins in their outer membranes. More distant relationships are seen between the Omp85 family and both the core proteins in two-partner secretion systems and the Toc75 family of protein translocases found in plastid outer envelopes. Aspects of the ancestry and molecular architecture of the Omp85 family of proteins is providing insight into the mechanism by which proteins might be integrated and assembled into bacterial outer membranes.     

Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain

beta-Barrel proteins are present in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The central component of their assembly machinery is called Omp85 in bacteria. Omp85 is predicted to consist of an integral membrane domain and an amino-terminal periplasmic extension containing five polypeptide-transport-associated (POTRA) domains. We have addressed the function of these domains by creating POTRA domain deletions in Omp85 of Neisseria meningitidis. Four POTRA domains could be deleted with only slight defects in Omp85 function. Only the most carboxy-terminal POTRA domain was essential, as was the membrane domain. Thus, similar to the mitochondrial Omp85 homologue, the functional core of bacterial Omp85 consists of its membrane domain and a single POTRA domain, that is, POTRA5.     

Aggregatibacter actinomycetemcomitans Omp29 is associated with bacterial entry to gingival epithelial cells by F-actin rearrangement

The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29(+)/OmpA(-) E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29(-)/OmpA(-) E. coli. While the entry of Aa and Omp29(+)/OmpA(-) E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.