Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.     

Identification of the sid outer membrane receptor protein in Salmonella typhimurium SL1027.

Summary A protein of molecular weight 78,000 daltons, missing in albomycin and phage ES18 resistant mutants, has been identified in the outer membrane of Salmonella typhimurium SL1027. Mutants with a tonB like resistance and overproduction of outer membrane proteins due to iron shortage were also isolated. The mutation which leads to the protein deficiency maps in the sid gene region, the mutation related to overproduction of proteins maps near trp. Although the S. typhimurium and the E. coli protein mediate translocation of the iron complex ferrichrome and the structurally analogous antibiotic albomycin through the outer membrane no cross-reactivity exists in binding the phages T5, T1 and ES18 or colicin M.     

Identification of immunodominant linear B-cell epitopes within the major outer membrane protein of Chlamydia trachomatis

Chlamydia trachomatis is one of the most prevalent sexually transmitted pathogens. Chlamydial major outer membrane protein (MOMP) can induce strong cellular and humoral immune responses in murine models and has been regarded as a potential vaccine candidate. In this report, the amino acid sequence of MOMP was analyzed using computer-assisted techniques to scan B-cell epitopes, and three possible linear B-cell epitopes peptides (VLKTDVNKE, TKDASIDYHE, TRLIDERAAH) with high predicted antigenicity and high conservation were investigated. The DNA coding region for each potential epitope was cloned into pET32a(+) and expressed as Trx-His-tag fusion proteins in Escherichia coli. The fusion proteins were purified by Ni-NTA agarose beads and followed by SDS-PAGE and western blot analysis. We immunized mice with these three fusion proteins. The sera containing anti-epitope antibodies from the immunized mice could recognize C. trachomatis serovars D and E in ELISA. Antisera of these fusion proteins displayed an inhibitory effect on invasion of serovar E by in vitro neutralization assays. In addition, serum samples from convalescent C. trachomatis-infected patients were reactive with the epitope fusion proteins by western blot assay. Our results showed that the epitope sequences selected by bioinformatic analysis are highly conserved C. trachomatis MOMP B-cell epitopes, and could be good candidates for the development of subunit vaccines, which can be used in clinical diagnosis.     

Crystallization of the immunodominant outer membrane protein OmpC; the first protein crystals from Salmonella typhi, a human pathogen.

OmpC, a surface antigen of Salmonella typhi was crystallized after several attempts, using PEG 3350. Well shaped hexagonal crystals were grown from vapor diffusion method using octyl glucoside and C12E9 as detergents. Crystals are sensitive to X-ray and diffract weakly up to 7 A. Porin isoforms, due to the bound lipopolysaccharides, could be the cause for poor diffraction. Crystal quality depends largely on the purification method, and in case of LPS contamination, the genetic background of the bacteria. Crystallization and initial data collection suggest optimum conditions and the method of choice for OmpC crystallization.     

Prediction and characterization of T-cell epitopes for epitope vaccine design from outer membrane protein of Neisseria meningitidis serogroup B

Neisseria meningitidis serogroup B (MC58) is a leading cause of meningitis and septicaemia, principally infects the infants and adolescents. No vaccine is available for the prevention of these infections because the serogroup B capsular polysaccharide is unable to stimulate an immune response, due to its similarity with polysialic acid. To overcome these obstacles, we proposed to develop a peptide based epitope vaccine from outer membrane protein contained in outer membrane vesicles (OMV) based on our computational analysis. In OMV a total of 236 proteins were identified, only 15 (6.4%) of which were predicted to be located in outer membrane. The major requirement is the identification and selection of T-cell epitopes that act as a vaccine target. We have selected 13 out of 15 outer membrane proteins from OMV proteins. Due to similarity of the fkpA and omp85 with the human FKBP2 and SAMM50 protein, we removed these two sequences from the analysis as their presence in the vaccine is likely to elicit an autoimmune response. ProPred and ProPred1 were used to predict promiscuous helper T Lymphocytes (HTL) and cytotoxic T Lymphocytes (CTL) epitopes and MHCPred for their binding affinity in N. meningitidis serogroup B (MC58), respectively. Binding peptides (epitopes) are distinguished from nonbinding peptides in properties such as amino acid preference on the basis of amino acid composition. By using this dataset, we compared physico-chemical and structural properties at amino acid level through amino acid composition, computed from ProtParam server. Results indicate that porA, porB, opc, rmpM, mtrE and nspA are more suitable vaccine candidates. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage.     

Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF

Abstract The outer membrane proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membraine protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with proteins from representatives of the rRNA homology group I of the Pseudomonadaceae . Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeuroginosa for phagocytosis. These epitopes were partially masked by lipopolysacharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved protein with several conserved surface antigenic epitopes.     

Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium.

A panel of monoclonal antibodies, seven against the trimeric and seven against the monomeric forms to outer membrane protein D (OmpD) of Salmonella typhimurium were produced. The specificities of these monoclonal antibodies for the porin proteins of S. typhimurium and their cross-reactions with Salmonella porins OmpC and OmpF were determined by Western immunoblotting and enzyme-linked immunosorbent assay. We observed that OmpD shared more epitopes and had greater structural similarity with OmpC than with OmpF.     

Major outer membrane protein in Salmonella typhimurium induced by maltose.

A maltose-induced major outer membrane protein (the 44K protein) is demonstrated in Salmonella typhimurium. This protein resembles the lambda receptor of Escherichia coli in its location, induction properties, apparent molecular weight, and association with the peptidoglycan layer of the cell wall. The 44K protein is missing in certain Salmonella Mal- mutants, which are also missing a protein analogous to the maltose-binding protein of E. coli. Thus, these mutants may be defective in the control of maltose genese in Salmonella. The proteins appear to be closely related, as indicated by cross-reaction of the Salmonella protein with the antiserum raised against the lambda receptor; however, they are not identical, since the peptide patterns obtained after limited proteolysis are completely different. Bacteriophage lambda does not use the 44K protein as a receptor.     

Accelerated microevolution in an outer membrane protein (OMP) of the intracellular bacteria <Emphasis Type=Italic>Wolbachia</Emphasis>

Background  

Outer membrane proteins (OMPs) of Gram-negative bacteria are key players in the biology of bacterial-host interactions. However, while considerable attention has been given to OMPs of vertebrate pathogens, relatively little is known about the role of these proteins in bacteria that primarily infect invertebrates. One such OMP is found in the intracellular bacteria Wolbachia, which are widespread symbionts of arthropods and filarial nematodes. Recent experimental studies have shown that the Wolbachia surface protein (WSP) can trigger host immune responses and control cell death programming in humans, suggesting a key role of WSP for establishment and persistence of the symbiosis in arthropods.     

Evaluation of recombinant outer membrane protein based vaccine against Salmonella Typhimurium in birds

Food-borne diseases caused by Salmonella enterica from poultry sources represent an important public health problem and no reliable control by vaccination has proved effective despite research. The aim of the present study was to evaluate the use of recombinant OmpC protein for immunization of birds to elucidate its protection against virulent Salmonella Typhimurium. The recombinant OmpC protein was prepared after cloning and expressing ompC gene and was characterized by SDS-PAGE and Western blot analyses. The protein preparations were tested as vaccine candidate in layer birds by comparing the immune response, protection and organ clearance against crude lysate and control. The biologically functional recombinant 43 kDa truncated OmpC protein proved to be a good immunogen which induced a significantly high humoral immune response than control. At the same time, it primed a stable cell-mediated immune response. A protective index (based on faecal shedding of organism) of rOmpC based preparations ranged between 50 and 75% as observed for 3 weeks after challenge. Therefore, the protein preparations conferred satisfactory protection against challenge infections with virulent strains of S. Typhimurium as evidenced by limited faecal shedding and minimal detection of Salmonella from edible tissues and eggs. These findings suggest the possibility to explore the use of S. enterica OMP protein for the production of novel vaccine.     

Immunofluorescent Identification of Salmonella typhi During a Typhoid Outbreak

Immunofluorescence and conventional bacteriological methods were compared for their ability to detect Salmonella typhi in 134 fecal specimens from 105 individuals associated with an outbreak of typhoid fever. Smears prepared from untreated fecal material (direct method) and after a preliminary incubation in selenite F broth (delayed method) were tested with an anti-Vi serum conjugated with fluorescein isothiocyanate. The delayed method was more sensitive than the direct method in detecting S. typhi. The delayed method was positive in 40 of 41 patients positive by culture methods, but gave positive or questionable reactions in 11 presumably uninfected individuals. The fluorescent-antibody test employing a Vi conjugate is a satisfactory screening procedure for detecting S. typhi, but all positives must be confirmed bacteriologically.     

Identification of MAGE-C1 (CT-7) epitopes for T-cell therapy of multiple myeloma

Multiple myeloma is incurable with standard therapies but is susceptible to a T-cell-mediated graft versus myeloma effect after allogeneic stem cell transplantation. We sought to identify myeloma-specific antigens that might be used for T-cell immunotherapy of myeloma. MAGE-C1 (CT-7) is a cancer-testis antigen that is expressed by tumor cells in >70% of myeloma patients and elicits a humoral response in up to 93% of patients with CT-7⁺ myeloma. No CD8⁺ T-cell epitopes have been described for CT-7, so we used a combination of reverse immunology and immunization of HLA-A2 transgenic mice with a novel cell-based vaccine to identify three immunogenic epitopes of CT-7 that are recognized by human CD8⁺ T-cells. CT-7-specific T-cells recognizing two of these peptides are able to recognize myeloma cells as well as CT-7 gene-transduced tumor cells, demonstrating that these epitopes are naturally processed and presented by tumor cells. This is the first report of the identification of immunogenic CD8⁺ T-cell epitopes of MAGE-C1 (CT-7), which is the most commonly expressed cancer-testis antigen found in myeloma, and these epitopes may be promising candidate targets for vaccination or T-cell therapy of myeloma or other CT-7⁺ malignancies.     

Identification of an iron-regulated,hemin-binding outer membrane protein in Sinorhizobium meliloti

Rhizobia are soil bacteria that are able to establish symbiotic associations with leguminous hosts. In iron-limited environments these bacteria can use iron present in heme or heme compounds (hemoglobin, leghemoglobin). Here we report the presence in Sinorhizobium meliloti of an iron-regulated outer membrane protein that is able to bind hemin but not hemoglobin. Protein assignment was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Tryptic peptides correlated with the mass measurements obtained accounted for 54% of the translated sequence of a putative heme receptor gene present in the chromosome of S. meliloti 1021. The results which we obtained suggest that this protein (designated ShmR for Sinorhizobium heme receptor) is involved in high-affinity heme-mediated iron transport.     

Restoration of membrane incorporation of an Escherichia coli outer membrane protein (OmpA) defective in membrane insertion

The mechanism of sorting, to the outer membrane, of the 325-residue Escherichia coli protein OmpA has been investigated. It is thought to traverse the membrane eight times in antiparallel beta-strands, forming an amphiphilic beta-barrel which encompasses residues 1 to about 170; the COOH-terminal moiety is periplasmic. A mutant, carrying the substitutions Leu164----Pro and Val166----Asp within the last beta-strand (residues 160-170), has been described which was unable to assemble in the membrane (Klose, M., MacIntyre, S., Schwarz, H., and Henning, U. (1988) J. Biol. Chem. 263, 13297-13302). Linkers were inserted between the codons for residues 164 and 165 of the mutant protein. Of 13 different genes recovered, five encoded proteins which had regained the ability to assemble in the membrane. The properties of the mutant proteins, together with a structure prediction method, indicate the following rules for the final beta-strand to be compatible with, or possibly initiate, membrane insertion: (i) it must be amphiphilic or hydrophobic while its primary structure as such is fairly unimportant, (ii) it must extend over at least 9 residues, and (iii) it must not contain a proline residue around its center. One of the genes recovered coded for OmpA up to residue 164 and then followed by 10 linker-encoded residues. This 174-residue polypeptide was assembled in the membrane but did not, in contrast to all other proteins, expose sites sensitive to trypsin at the inner face of the membrane. This behavior agrees perfectly well with the OmpA model.     

Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi

In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T _m?~?69 °C at pH 7.0, monitored by change in MRE_{222 nm}). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C _m (~1 M), while urea is characterized by high C _m (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.     

Infection-induced antibodies against the major outer membrane protein of Campylobacter jejuni mainly recognize conformational epitopes

The major outer membrane protein (MOMP) of Campylobacter jejuni is an abundant surface protein with a pore-forming function and may be a potential candidate for vaccine development. Despite the fact that MOMP is immunogenic and the recombinant MOMP (rMOMP) can be readily produced in Escherichia coli, the nature of the antibody response to MOMP during in vivo infection is not well understood. In this study, various methods involving detergent replacement and liposome reconstitution were used to refold rMOMP, and antibody responses to MOMP elicited in Campylobacter-colonized chickens were evaluated using sera from chickens either naturally or experimentally infected by C. jejuni. The results demonstrated that proteoliposomes restored the reactivity of rMOMP to rabbit antibodies elicited by native MOMP, indicating the recovery of native MOMP conformation by this refolding method. Importantly, sera from naturally or experimentally infected chickens reacted weakly with denatured rMOMP, but strongly with rMOMP reconstituted in proteoliposome, suggesting that the chicken antibody response to MOMP is predominantly directed against conformational epitopes. These observations provide direct evidence for conformation-dependent humoral responses to MOMP induced by Campylobacter infection, demonstrate that C. jejuni MOMP is immunogenic in its natural host and suggest that proteoliposomes may be potentially used for the evaluation of rMOMP-based vaccines.     

Demonstration of an antigenic protein specific for Salmonella typhi

Current studies were undertaken to determine the presence of a specific antigenic protein on the outer membrane of Salmonella typhi. Immunoblot analysis using sera from patients with fevers revealed that the 50 kD band was specifically recognized only by typhoid sera. The 50 kD band located on the outer membrane is protein by nature and is not a Vi (capsular), dH (flagellar), or O9 (somatic) antigen of S. typhi. These results indicate the usefulness of the specific antigen in the development of a serodiagnostic test for typhoid fever since antibodies of both the IgM and IgG class responses were obtained.