Prevalence of outer membrane porin alteration in beta-lactam-antibiotic-resistant Enterobacter aerogenes

We evaluated the prevalence of impermeability as a mechanism associated with resistance against beta-lactam antibiotics in members of the family Enterobacteriaceae. During a 1-year period, 80 strains were selected from 3,110 routinely isolated strains according to their noticeable cross-resistance pattern to cephalosporins. They were tested for (i) outer membrane nonspecific porins involved in the entry of small hydrophilic molecules; (ii) the MICs of cefepime, cefotaxime, imipenem, and moxalactam; and (iii) beta-lactamase production. Immunological investigations using specific probes showed that 23 of 80 strains presented an alteration of the porin content, most of them expressing an additional resistance mechanism. The prevalence of this porin-deficient phenotype is especially high in Enterobacter aerogenes and concerns 6.4% of the clinical isolates.     

Computer simulations of the OmpF porin from the outer membrane of Escherichia coli

Molecular dynamics simulations were used to study the structure and dynamics of the Escherichia coli OmpF porin, which is composed of three identical 16-stranded beta-barrels. Simulations of the full trimer in the absence of water and the membrane led to significant contraction of the channel in the interior of each beta-barrel. With very weak harmonic constraints (0.005 kcal/mol A2/atom) applied to the main-chain C alpha atoms of the beta-barrel, the structure was stabilized without alteration of the average fluctuations. The resulting distribution of the fluctuations (small for beta-strands, large for loops and turns) is in good agreement with the x-ray B factors. Dynamic cross-correlation functions showed the importance of coupling between the loop motions and barrel flexibility. This was confirmed by the application of constraints corresponding to the observed temperature factors to the barrel C alpha atoms. With these constraints, the beta-barrel fluctuations were much smaller than the experimental values because of the intrinsic restrictions on the atomic motions, and the loop motions were reduced significantly. This result indicates that considerable care is required in introducing constraints to keep proteins close to the experimental structure during simulations, as has been done in several recent studies. Loop 3, which is thought to be important in gating the pore, undergoes a displacement that shifts it away from the x-ray structure. Analysis shows that this arises from the breakdown of a hydrogen bond network, which appears to result more from the absence of solvent that from the use of standard ionization states for the side chains of certain beta-barrel residues.     

Antigen-specific T-cell responses in humans after intranasal immunization with a meningococcal serogroup B outer membrane vesicle vaccine

We have studied the ability of the Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine, when administered intranasally without adjuvant, to induce T-cell responses in humans. A group of 12 vaccinees was immunized with four doses of OMVs (250 micrograms of protein/dose) at weekly intervals, and a single booster dose was given 5 months later. In vitro T-cell proliferation in response to the OMV vaccine, purified PorA (class 1) protein, PorB (class 3) protein, and one unrelated control antigen (Mycobacterium bovis BCG) was measured by [3H]thymidine incorporation into peripheral blood mononuclear cells obtained from the vaccinees before and after the immunizations. The nasal OMV immunizations induced antigen-specific T-cell responses in the majority of the vaccinees when tested against OMVs (7 of 12) and the PorA antigen (11 of 12). None of the vaccinees showed a vaccine-induced T-cell response to the PorB antigen after the initial four doses. Although some individuals responded to all the vaccine antigens after the booster dose, this response was not significant when the vaccinees were analyzed as a group. We have also demonstrated that the PorA antigen-specific T-cell responses correlated with anti-OMV immunoglobulin A (IgA) levels in nasal secretions, with anti-OMV IgG levels in serum, and with serum bactericidal activity. In conclusion, we have shown that it is possible to induce antigen-specific T-cell responses in humans by intranasal administration of a meningococcal OMV vaccine without adjuvant.     

Participation of the lungs in the formation of an immune response

The process of accumulation of the antibody-forming cells (AFC) in the lungs after vaccination of rabbits with the typhoid antigen was studied. No AFC was revealed in the lungs after single aerosol and subcutaneous immunization; double administration of the antigen through the respiratory tracts was followed by significant AFC accumulation in the lung tissue; individual antibody-synthesizing cells were found in the lung after subcutaneous application. Revaccination was followed by intensification of the immune response of the lung in both methods of vaccination; the content of antibody-producing cells in the aerosol-immunized rabbits was much greater than in the animals vaccinated subcutaneously. This indicated a definite role of the lungs in the immunogenesis in the case of the antigen administration through the respiratory tract.     

Expression cloning and humoral immune response to the nucleocapsid and membrane proteins of equine arteritis virus

To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M). The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV. Some horse sera, however, showed immune reactivity to the MBP fusion partner protein. Based on a total of 32 horse sera analyzed for the presence of EAV antibodies by immunoblot, using the MBP-N or -M fusion proteins and the Xa factor cleavage EAV products, and in the serum neutralization test, there was 100% concordance between the assays. Sera from horses experimentally infected with EAV were reactive in the immunoblot test with both the MBP-N and the MBP-M fusion proteins by day 14 after EAV exposure. The reactivity continued to the end of the experiment at day 145 after infection. This immune reactivity correlated with the detection of neutralizing antibodies in the serum samples. Based on these findings, the recombinant N and M proteins might be useful for serodetection of EAV-infected animals.     

Cytomegaloviruses use multiple mechanisms to elude the host immune response

The study of the effects of cytomegaloviruses on the MHC class I-restricted antigen presentation pathway has yielded an embarrassment of riches. The human cytomegalovirus (HCMV) encodes at least five to six different glycoproteins, each interfering in a different way with elimination of the virus by the host immune system. Most likely, it is the concerted action of these glycoproteins that allows HCMV to escape from elimination by the host immune system during acute and perhaps also persistent infection. Prime targets of these CMV glycoproteins are MHC class I glycoproteins: the very molecules that signal the presence of a virally infected cell to the immune system. Recently, several novel links in the multi-step process of immune evasion by HCMV have been discovered.     

SurA assists the folding of Escherichia coli outer membrane proteins

Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.     

Antibody response to outer membrane protein of nontypeable Haemophilus influenzae in otitis-prone children

One of the major outer membrane proteins of nontypeable Haemophilus influenzae, P6, is highly conserved among strains, serves as a target for bactericidal antibody, and has been proposed as a possible vaccine candidate. The serum antibody response to P6 was studied in otitis-prone and normal children by an enzyme-linked immunosorbent assay. Of 20 otitis-prone children, 12 (60%) had a serum IgG antibody response to P6 after otitis media; however, the mean acute antibody level for the group, 4.6 micrograms/ml, was not significantly different from the convalescent level, 5.4 micrograms/ml. Anti-P6 antibody levels were also measured longitudinally for 10 to 25 months in 30 otitis-prone and 13 healthy children. Antibody levels increased sevenfold in the normal group compared with less than three-fold for the otitis-prone group and were significantly higher in the normal children after the age of 18 months (p < 0.05). Finally, otitis-prone children who had two or more episodes of otitis media with nontypeable H. influenzae did not have an anamnestic antibody response to P6. The failure to recognize P6 as a specific immunogen may account for recurrent infections. Moreover, the data suggest that otitis-prone children may not respond adequately to a vaccine containing P6.     

The characteristics of the immune response in acute viral hepatitis B

The clinical and pathogenetic importance of a number of features characterizing cell-mediated immunity and nonspecific protective factors in acute virus hepatitis B. 124 patients with hepatitis B virus (HBV) infection were placed under observation. Of these, 115 patients had acute virus hepatitis B, 6 patients had acute virus hepatitis of mixed etiology (B + delta) and 3 patients had chronic virus hepatitis B. The study included, besides the detection of virus hepatitis markers and the biochemical analysis of blood, the determination of subpopulations of peripheral blood lymphocytes (CD3, CD4, CD8, CD57), the functional activity of natural killers, characteristics of the interferon status, serum neopterin and beta 2-microglobulin in blood serum. Considerable changes in cell-mediated immunity and the interferon system were found to occur and the optimum immune response in acute virus hepatitis B was characterized.     

Antibody responses of cattle to outer membrane proteins of Pasteurella multocida A:3

OBJECTIVE: To quantify the serum antibody responses to Pasteurella multocida A:3 outer membrane proteins (OMP) for cattle vaccinated with the homologous serogroup and to correlate those responses with the extent of experimentally induced pneumonia. ANIMALS: 29, 5- to 8-month-old beef-type calves. PROCEDURE: Calves were vaccinated SC or by aerosal exposure on days 0 and 7 with live or killed P multocida or phosphate-buffered saline solution (control) and subsequently challenge exposed with virulent P multocida. Antibody responses to P multocida A:3 outer membranes were quantified, using an ELISA. Antibody responses to individual OMP were detected by immunoblot analysis (western blot) and were quantified by densitometry. Antibody responses were compared among groups of calves and for various times after vaccination. Regression analyses were used to determine whether significant correlations existed between lesion scores and antibody responses to either whole outer membranes or to individual OMP. RESULTS: By ELISA, antibody responses to outer membranes for calves aerosol vaccinated with live P multocida were significantly (P < 0.05) greater than those for control calves or for killed P multocida vaccinates. There was a significant (P < 0.05) correlation between lesion score and antibody responses to outer membranes. By western blotting and densitometry, antibodies to 11 prominent OMP (100, 97, 90, 85, 74, 53, 46, 35, 32, 21, and 16 kd) were identified and quantified. In experiment 1, SC vaccination with live P multocida increased antibody binding to all protein bands except 85-, 74-, and 35-kd bands. Aerosol vaccination with live P multocida stimulated increases in antibody binding to all bands except 100 and 16 kd. Antibody responses to the 97-, 90-, 74-, and 35- kd bands were significantly (P < 0.05; greater for live aerosol vaccinates than for control calves. In experiment 2, antibody responses were not different between the killed P multocida vaccinates or control calves Antibody responses for live P multocida aerosol vaccinates were significantly (P < 0.05) greater than those for control calves for the 100-, 90-, 85-, 74-, 53-, 35-, and 16-kd bands. Regression analyses indicated significant correlations (P < 0.05) between lesion score and antibody responses to the 100-, 90-, 53-, 46-, 35-, and 32-kd OMP. CONCLUSIONS: Several OMP of P multocida type A:3 may be important for stimulating immunity to the organism in cattle.     

Extracellular matrix proteins as regulators of the immune response

We describe a method for construction of hymeric bacteriophage T4 particles displaying foreign polypeptides on their surface. The method is based on our finding that minor T4 fibrous protein fibritin encoded by gene wac (whisker's antigen control) could be lengthened at the C terminus without impairing its folding or binding to the phage particle. The lengthened fibritin gene could easily be transferred into the T4 genome by homologous recombination with a plasmid containing the modified gene wac. The modified gene wac is expressed properly during phage reproduction, and the lengthened fibritin is bound to phage particles. As an example of this type of method, we have obtained the hymeric T4 particles carrying a polypeptide of 53 residues, 45 of which are from the pre-S2 region of hepatitis B virus. The T4 display vector extends currently available display systems.     

Secretory immune response to membrane antigens during Giardia lamblia infection in humans

The secretory immune response in humans infected with Giardia lamblia was studied by using saliva samples and a membrane-rich protein fraction. The membrane fraction, studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed 24 antigen bands, ranging from 170 to 14 kDa. Saliva samples from giardiasis patients showed a heterogeneous response against the membrane fraction when they were assayed by immunoblotting. Among the antigens recognized by patient saliva samples, those of 170, 105, 92, 66, 32, 29, and 14 kDa stood out. These antigens were not recognized by saliva samples from healthy individuals. They may be of importance in future studies of protection from or diagnosis of G. lamblia infections.     

Outer membrane protein B1, an iron-repressible protein conserved in the outer membrane of Moraxella (Branhamella) catarrhalis, binds human transferrin

Moraxella (Branhamella) catarrhalis is a gram-negative human mucosal pathogen, which primarily causes otitis media in young children. However, this bacterium is also a common cause of lower respiratory tract infections in adults with underlying lung disease. Our previous data have shown that M. catarrhalis expresses iron-repressible outer membrane proteins in response to iron limitation. We have extended these observations to demonstrate that one of these proteins, termed outer membrane protein (OMP) B1, binds human transferrin. Using a newly developed monoclonal antibody to OMP B1, we determined that this protein is conserved in the iron-stressed outer membranes of all clinical isolates of M. catarrhalis tested to date. Furthermore, our data have confirmed that children infected with M. catarrhalis have immunoglobulin G antibodies to OMP B1 in their convalescent sera. These current data suggest that OMP B1 is immunogenic and expressed in vivo and may be involved in an iron uptake mechanism utilized by M. catarrhalis.     

Recent advances in the large scale fermentation of Neisseria meningitidis group B for the production of an outer membrane protein complex

BACKGROUND: As women with cystic fibrosis are living longer, pregnancy is becoming increasingly common. The combined experience of pregnancies in women with cystic fibrosis from adult centres in the Midlands and North of England has been examined. METHODS: A retrospective study of the case notes of 22 pregnancies in 20 patients with cystic fibrosis examined changes in lung function, body weight, and microbiological status during the course of pregnancy. Duration of pregnancy, birth weight, and maternal survival were amongst other variables studied. The relation between values before pregnancy and important outcome measures were examined. RESULTS: Eighteen of 22 pregnancies were completed producing healthy, non-cystic fibrosis infants (12 female). Mothers lost 13% of FEV1 and 11% of FVC during pregnancy, most of which was regained. Body weight changes were variable, but most mothers gained weight (mean weight gain 5.7 kg). Microbiological status remained unchanged. Six infants were preterm and two were light for dates. Four mothers died up to 3.2 years following delivery. Of the prepregnancy parameters examined, %FEV1 showed the best correlation with maternal weight gain, gestation, birth weight, and maternal survival. CONCLUSIONS: Pregnancy was well tolerated by most mothers with cystic fibrosis although those with moderate to severe lung disease (%FEV1 < 60%) before pregnancy fared worse, producing preterm infants and suffering increased loss of lung function and mortality compared with mildly affected mothers. Prepregnancy %FEV1 appears to be the most useful predictor of important outcome measures in pregnancies in women with cystic fibrosis.     

Molecular characterization of an outer membrane protein of Actinobacillus actinomycetemcomitans belonging to the OmpA family

The major outer membrane protein (OMP) of Actinobacillus actinomycetemcomitans is an OmpA homolog that demonstrates electrophoretic heat modifiability. The gene encoding this protein was isolated from a genomic library of A. actinomycetemcomitans NCTC 9710 by immunoscreening with serum from a patient with localized juvenile periodontitis. Expression of the cloned gene in Escherichia coli and subsequent Western blot analysis revealed a protein with an approximate molecular mass of 34 kDa. The amino acid sequence predicted from the cloned gene demonstrated that the mature protein had a molecular mass of 34,911 Da and significant identity to members of the OmpA family of proteins. We have named the major OMP of A. actinomycetemcomitans Omp34, and its corresponding gene has been named omp34.     

Affinity of the periplasmic chaperone Skp of Escherichia coli for phospholipids, lipopolysaccharides and non-native outer membrane proteins. Role of Skp in the biogenesis of outer membrane protein

The Skp protein of Escherichia coli has been proposed to be a periplasmic molecular chaperone involved in the biogenesis of outer membrane proteins. In this study, evidence is obtained that Skp exists in two different states characterized by their different sensitivity to proteases. The conversion between these states can be modulated in vitro by phospholipids, lipopolysaccharides and bivalent cations. Skp is able to associate with and insert into phospholipid membranes in vitro, indicating that it may associate with phospholipids in the inner and/or outer membrane in vivo. In addition, it interacts specifically with outer membrane proteins that are in their non-native state. We propose that Skp is required in vivo for the efficient targeting of unfolded outer membrane proteins to the membrane.     

The maturation of the immune response

In a T-cell dependent immune response, the repertoire of antigen-activated B cells is diversified by a hypermutation mechanism. Only high-affinity variants are selected into the pool of memory cells. This maturation process takes place in a special micro-environment, the germinal centre. Here, Claudia Berek and Mike Ziegner discuss the mechanisms underlying these processes.