Analysis of major virulence factors in Porphyromonas gingivalis under various culture temperatures using specific antibodies

Porphyromonas gingivalis is implicated in the occurrence of adult periodontitis. We have previously identified major outer membrane proteins from P. gingivalis, which include representative virulence factors such as gingipains, a 75 kDa major protein, RagA, RagB, and putative porin. Fimbriae, another important virulence factor, exist on the cell surface. In this study, we identified major supernatant proteins. They were fimbrilin, the 75 kDa major protein, gingipains and their adhesin domains. Microscopic examination showed that supernatant proteins formed vesicle-like and fimbrial structures. To learn more about the character of this bacterium, we examined effects of growth temperature on localization and expression of these virulence factors. In general, localization of major virulence factors did not change at the various growth temperatures used. Most of the 75 kDa major protein, RagA, RagB, and putative porin were found in the envelope fraction, not in cell-free culture supernatant. Gingipains were found in both the envelope fraction and supernatant. More than 80% of fimbriae were associated with cells, less than 20% migrated to the supernatant. Most fimbriae existed in the whole cell lysate, although there was a small amount in the envelope fraction. When the growth temperature was increased, expression of fimbriae, gingipains, the 75 kDa major protein, RagA, and RagB decreased. However, temperature had almost no effect on expression of putative porin. The tendency for expression of major virulence factors to decrease at higher temperatures may enable P. gingivalis to survive under hostile conditions.     

Tobacco-induced alterations to Porphyromonas gingivalis–host interactions

Smokers are more susceptible than non-smokers to persistent infection by Porphyromonas gingivalis , a causative agent of periodontitis. Patients who smoke exhibit increased susceptibility to periodontitis and are more likely to display severe disease and be refractory to treatment. Paradoxically, smokers demonstrate reduced clinical inflammation. We show that P. gingivalis cells exposed to cigarette smoke extract (CSE) induce a lower proinflammatory response (tumour necrosis factor-α, interleukin-6, interleukin-12 p40) from monocytes and peripheral blood mononuclear cells than do unexposed bacteria. This effect is reversed when CSE-exposed bacteria are subcultured in fresh medium without CSE. Using microarrays representative of the P. gingivalis genome, CSE-exposure resulted in differential regulation of 6.8% of P. gingivalis genes, including detoxification and oxidative stress-related genes; DNA repair genes; and multiple genes related to P. gingivalis virulence, including genes in the major fimbrial and capsular operons. Exposure to CSE also altered the expression of outer membrane proteins, most notably by inducing the virulence factors RagA and RagB, and a putative lipoprotein cotranscribed with the minor fimbrial antigen. Therefore, CSE represents an environmental stress to which P. gingivalis adapts by altering gene expression and outer membrane proteins. These changes may explain, in part, the altered virulence and host–pathogen interactions that have been documented in vivo in smokers with periodontal disease.     

High‐resolution architecture of the outer membrane of the Gram‐negative bacteria Roseobacter denitrificans

The outer membrane of Gram‐negative bacteria protects the cell against bactericidal substances. Passage of nutrients and waste is assured by outer membrane porins, beta‐barrel transmembrane channels. While atomic structures of several porins have been solved, so far little is known on the supramolecular structure of the outer membrane. Here we present the first high‐resolution view of a bacterial outer membrane gently purified maintaining remnants of peptidoglycan on the perisplasmic surface. Atomic force microscope images of outer membrane fragments of the size of ～50% of the bacterial envelope revealed that outer membrane porins are by far more densely packed than previously assumed. Indeed the outer membrane is a molecular sieve rather than a membrane. Porins cover ～70% of the membrane surface and form locally regular lattices. The potential role of exposed aromatic residues in the formation of the supramolecular assembly is discussed. Finally, we present first structural data of the outer membrane porin from the marine Gram‐negative bacteria Roseobacter denitrificans, and we perform a sequence alignment with porins of known structure.     

Effects of various growth conditions in a chemostat on expression of virulence factors in Porphyromonas gingivalis

Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide-limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.     

Structure of the OmpA-like domain of RmpM from Neisseria meningitidis

RmpM is a putative peptidoglycan binding protein from Neisseria meningitidis that has been shown to interact with integral outer membrane proteins such as porins and TonB-dependent transporters. Here we report the 1.9 A crystal structure of the C-terminal domain of RmpM. The 150-residue domain adopts a betaalphabetaalphabetabeta fold, as first identified in Bacillus subtilis chorismate mutase. The C-terminal RmpM domain is homologous to the periplasmic, C-terminal domain of Escherichia coli OmpA; these domains are thought to be responsible for non-covalent interactions with peptidoglycan. From the structure of the OmpA-like domain of RmpM, we suggest a putative peptidoglycan binding site and identify residues that may be essential for binding. Both the crystal structure and solution experiments indicate that RmpM may exist as a dimer. This would promote more efficient peptidoglycan binding, by allowing RmpM to interact simultaneously with two glycan chains through its C-terminal, OmpA-like binding domain, while its (structurally uncharacterized) N-terminal domain could stabilize oligomers of porins and TonB-dependent transporters in the outer membrane.     

Isolation of outer membrane of Synechocystis sp. PCC 6803 and its proteomic characterization

In this report, we describe a newly developed method for isolating outer membranes from Synechocystis sp. PCC 6803 cells. The purity of the outer membrane fraction was verified by immunoblot analysis using antibodies against membrane-specific marker proteins. We investigated the protein composition of the outer membrane using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by database identification. Forty-nine proteins were identified corresponding to 29 different gene products. All of the identified proteins have a putative N-terminal signal peptide. About 40% of the proteins identified represent hypothetical proteins with unknown function. Among the proteins identified are a Toc75 homologue, a protein that was initially found in the outer envelope of chloroplasts in pea, as well as TolC, putative porins, and a pilus protein. Other proteins identified include ABC transporters and GumB, which has a suggested function in carbohydrate export. A number of proteases such as HtrA were also found in the outer membrane of Synechocystis sp. PCC 6803.     

Crystal Structure of the Gtr1pGTP-Gtr2pGDP Protein Complex Reveals Large Structural Rearrangements Triggered by GTP-to-GDP Conversion

The heterodimeric Rag GTPases consisting of RagA (or RagB) and RagC (or RagD) are the key regulator activating the target of rapamycin complex 1 (TORC1) in response to the level of amino acids. The heterodimer between GTP-loaded RagA/B and GDP-loaded RagC/D is the most active form that binds Raptor and leads to the activation of TORC1. Here, we present the crystal structure of Gtr1p(GTP)-Gtr2p(GDP), the active yeast Rag GTPase heterodimer. The structure reveals that GTP-to-GDP conversion on Gtr2p results in a large conformational transition of this subunit, including a large scale rearrangement of a long segment whose corresponding region in RagA is involved in binding to Raptor. In addition, the two GTPase domains of the heterodimer are brought to contact with each other, but without causing any conformational change of the Gtr1p subunit. These features explain how the nucleotide-bound statuses of the two GTPases subunits switch the Raptor binding affinity on and off.     

Membrane-derived oligosaccharides (MDO's) promote closing of an E. coli porin channel.

The outer membrane of Escherichia coli is a diffusion barrier for macromolecules, but allows the passage of small hydrophilic solutes through non-specific channels, the porins. Some electrophysiological studies find reconstituted porins in a mostly open state, while those done with the patch-clamp technique performed on live cells suggest that the vast majority of the native channels are closed. We present here current measurements through porins from reconstituted outer membrane, which demonstrate that bacterial metabolites, the MDO's, which bathe the periplasmic side of the outer membrane, induce the channels to close. These findings illustrate that the degree of openness of porins can be regulated by compounds naturally found in bacteria.     

Effects of Various Growth Conditions in a Chemostat on Expression of Virulence Factors in Porphyromonas gingivalis

Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide-limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.     

Outer membrane of gram-negative bacteria. XVIII. Electron microscopic studies on porin insertion sites and growth of cell surface of Salmonella typhimurium.

Salmonella typhimurium contains three "major proteins" or "porins" (34K, 35K, and 36K) in the outer membrane. A mutant strain producing only the 35K porin was first grown in media containing high concentrations of NaCl to "repress" the porin synthesis and then was shifted into a medium without NaCl. The newly made porin molecules were then labeled with the ferritin-coupled antibody at various times after the shift, and the samples were examined by whole-mount, freeze-etching, and thin-section electron microscopy. These experiments showed that newly inserted porins appeared as discrete patches uniformly distributed over the surface of the cell and, furthermore, that the sites of adhesion between the inner and outer membrane were most probably the pathway by which the newly made porin molecules appeared on cell surface. The 34K and 36K porins were also inserted in the same manner, since the appearance of new porins at discrete sites all over the cell surface was also observed when cells with wild-type porin phenotype were treated with unlabeled antibody to block existing antigenic sites, subsequently regrown, and labeled with the ferritin-coupled antibody. Since porins comprise a major portion of the densely packed, relatively immobile, "protein framework" of the outer membrane, these results lead us to conclude that the outer membrane grows predominantly by diffuse intercalation rather than by the zonal growth mechanism.     

Reduced expression of outer-membrane proteins in beta-lactam-resistant mutants of Enterobacter cloacae

Two antibiotic-resistant mutants of Enterobacter cloacae (AZT-R and AMA-R), obtained by selection with aztreonam and carumonam, were studied. Both mutants were resistant to beta-lactam antibiotics. In addition, AMA-R was also resistant to chloramphenicol, trimethoprim and brodimoprim, whereas AZT-R was hypersensitive to these compounds. Cytoplasmic and outer membranes of these bacteria were separated by sucrose density gradient centrifugation. Analysis of the outer membranes using SDS-PAGE showed marked changes in the bands corresponding to the porins (between 35 and 40 kDa). In the two mutants, the 39 kDa band was reduced to approximately 30% of the wild-type and the 36.5 kDa band was absent. Labelling of the outer membranes with the hydrophobic photolabel 3-(trifluoomethyl)- 3-(m-[125I]iodophenyl)diazirine ([125I]TID) enabled the above bands as well as a 28.8 kDa band to be identified as integral membrane proteins, thus supporting the suggestion that they correspond to porins and OmpA protein, respectively. Whereas the changes observed in outer-membrane proteins are assumed to be responsible for resistance to beta-lactam antibiotics, the basis of hypersensitivity of AZT-R to hydrophobic antibiotics remains to be more clearly defined.     

Identification of outer membrane proteins of Yersinia pestis through biotinylation

The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with biotin. Tagged proteins were visualised through streptavidin probing of Western blots. Seven biotinylated proteins of Y. pestis were identified including two porins and the putative virulence factor catalase peroxidase.     

Acquisition of apparently intact and unmodified lipopolysaccharides from Escherichia coli by Bdellovibrio bacteriovorus.

The ability of Bdellovibrio bacteriovorus to relocalize the OmpF major outer membrane porins from its Escherichia coli prey to its own outer membranes is diminished in prey expressing smooth lipopolysaccharide (S-LPS). Since porins exist in the membrane complexed with LPS, we examined the LPS associated with relocalized porin to determine whether it had been acquired intact, mixed or replaced with Bdellovibrio LPS, or derivatized by the bdellovibrios. The relocalized trimers were found associated with the same LPS originally bound to them in the E. coli. The bulk-phase LPS from bdellovibrios grown on various chemotypes of rough prey was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine whether more than the trimer-bound LPS was acquired by the bdellovibrios. This analysis revealed bands of Bdellovibrio LPS matching the LPS chemotype of the prey. One or two other bands were identical in migration to the LPS of prey-independent mutants of B. bacteriovorus and represented bdellovibrio-synthesized LPS. The LPS of bdellovibrios grown on prey with radiolabeled lipid A showed radioactivity only in gel band positions identical with those of the prey's LPS. The amount of this prey-derived LPS was shown by enzyme-linked immunosorbent assay to reach a constant value during the purification of the bdellovibrios, and it represented approximately 25% of the total Bdellovibrio LPS. Immunoelectron microscopy confirmed the presence of prey-derived LPS on the cell surface of bdellovibrios, and no evidence could be found for bdellovibrio-induced modifications of the relocalized prey LPS.     

Identification of three porins in the outer membrane of Bacteroides fragilis

Abstract Four outer membrane proteins were purified to homogeneity from isolated outer membranes of Bacteroides fragilis ; three ( M _r 51000, 92000 and 125 000) had pore-forming activity in reconstituted liposomes as determined by swelling assay. Membrane vesicles containing the M _rmr 55 000 outer membrane protein showed no detectable pore-forming activity. The three B. fragilis porins formed pores that allowed the penetration of uncharged saccharides of M _r lower than 340–400, even though the efficiency of solute diffusion showed slight differences. The diffusion rates of glucose through the porins appeared to be lower than those through Escherichia coli porins.     

Borrelia burgdorferi BBA74, a periplasmic protein associated with the outer membrane, lacks porin-like properties

The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% alpha-helix with little beta-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins.     

Role of porins in iron uptake by Mycobacterium smegmatis

Many bacteria rely on siderophores to extract iron from the environment. However, acquisition of iron-loaded siderophores is dependent on high-affinity uptake systems that are not produced under high-iron conditions. The fact that bacteria are able to maintain iron homeostasis in the absence of siderophores indicates that alternative iron acquisition systems exist. It has been speculated that such low-affinity uptake of iron in Gram-negative bacteria includes diffusion of iron ions or chelates across the outer membrane through porins. The outer membrane of the saprophytic Mycobacterium smegmatis contains the Msp family of porins, which enable the diffusion of small and hydrophilic solutes, such as monosaccharides, amino acids, and phosphate. However, it is unknown how cations cross the outer membrane of mycobacteria. Here, we show that the Msp porins of M. smegmatis are involved in the acquisition of soluble iron under high-iron conditions. Uptake of ferric ions by a triple porin mutant was reduced compared to wild-type (wt) M. smegmatis. An intracellular iron reporter indicated that derepression of iron-responsive genes occurs at higher iron concentrations in the porin mutant. This was consistent with the finding that the porin mutant produced more siderophores under low-iron conditions than wt M. smegmatis. In contrast, uptake of the exochelin MS, the main siderophore of M. smegmatis, was not affected by the lack of porins, indicating that a specific outer membrane siderophore receptor exists. These results provide, to our knowledge, the first experimental evidence that general porins are indeed the outer membrane conduit of low-affinity iron acquisition systems in bacteria.     

TolA central domain interacts with Escherichia coli porins.

TolA is an inner membrane protein with three domains: a transmembrane N-terminus and periplasmic central and C-terminal domains. The interaction of TolA with outer membrane porins of Escherichia coli was investigated. Western blot analyses of cell extracts with anti-TolA antibodies indicated that TolA forms high molecular weight complexes specifically with trimeric OmpF, OmpC, PhoE and LamB, but not with OmpA. The interaction of purified TolA domains with purified porins was also studied. TolA interacted with OmpF, PhoE and LamB porins via its central domain, but not with either their denatured monomeric forms or OmpA. Moreover, the presence or absence of lipopolysaccharides associated with trimeric porins did not modify the interactions. These results suggest that the specific interaction of TolA with outer membrane porins might be relevant to the function of Tol proteins.     

Composition of major outer membrane proteins of Vibrio vulnificus isolates: effect of different growth media and iron deficiency

The outer membrane proteins of Vibrio vulnificus including isolates from humans, seawater and an asari clam were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A major outer membrane protein with an apparent molecular weight of 48,000 (48K protein) was common to all the strains grown in 3% NaCl-nutrient broth; however this 48K protein was not produced in any of the strains grown in chemically defined medium. Other major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 varied in number, relative amount and molecular weight depending on the strain. One to three new outer membrane proteins with molecular weights ranging from 74,000 to 85,000 were produced in the cells grown in iron-deficient medium. The 48K protein and one or two major proteins with molecular weights ranging from 35,000 to 37,000 in the cells grown in 3% NaCl-nutrient broth were not solubilized by 2% SDS at 60 C for 30 min and were resistant to trypsin, indicating that they are porins. On the other hand, in cells grown in chemically defined medium, one or two major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 might be porins.     

OmpW of Caulobacter crescentus Functions as an Outer Membrane Channel for Cations

Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures with the model of OmpW of C. crescentus suggested that it has a more hydrophilic interior and possibly a larger diameter.     

The periplasmic molecular chaperone protein SurA binds a peptide motif that is characteristic of integral outer membrane proteins

The Escherichia coli SurA protein is a periplasmic molecular chaperone that facilitates correct folding of outer membrane porins. The peptide binding specificity of SurA has been characterized using phage display of heptameric peptides of random sequence. The consensus binding pattern of aromatic-polar-aromatic-nonpolar-proline amino acids emerges for both SurA and a SurA "core domain," which remains after deletion of a peripheral peptidyl-proline isomerase domain. Isothermal titration calorimetry with a high affinity heptameric peptide of sequence WEYIPNV yields peptide affinities in the range of 1-14 microm for both SurA and its core domain. Although the peptide consensus aromatic-polar-aromatic-nonpolar-proline occurs infrequently in E. coli proteins, the less restrictive tripeptide motif aromatic-random-aromatic appears with greater-than-random frequency in outer membrane proteins and is prevalent in the "aromatic bands" of the porin beta barrel structures. Thus, SurA recognizes a peptide motif that is characteristic of integral outer membrane proteins.