Common epitopes of protein antigens in Neisseria and Moraxella

Cross reactions between N. meningitidis and M. catarrhalis proteins were studied with the use of a panel of monoclonal antibodies to M. catarrhalis protein antigens. All antigenic preparations under study were shown to give cross reactions between N. meningitidis serotype porin of 39 kD (strain B125) and M. catarrhalis proteins of 40-41 kD. These M. catarrhalis proteins belonged to main proteins of class F and had the function of porins in the cell. In addition, the epitope of 41-kD antigen, detected by monoclonal antibodies 3E10, is common for both N. meningitidis porin and N. meningitidis iron-regulated proteins of 70 and 50 kD. The epitope of M. catarrhalis protein of 67 kD, detected by monoclonal antibodies 1G6, is common for N. meningitidis porin and N. meningitidis iron-regulated proteins of 50 and 55 kD.     

Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.     

Rapid typing of Moraxella catarrhalis subpopulations based on outer membrane proteins using mass spectrometry

Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract both in children and in adults. Two subpopulations of this organism have been described that differ in 16S rRNA gene sequence and virulence traits. Three 16S rRNA types have been defined. 2-DE followed by protein identification by MS revealed significant differences in the outer membrane protein (OMP) patterns of each M. catarrhalis 16S rRNA type. Approximately 130 features were detected on the 2-DE map of each M. catarrhalis 16S rRNA type. However, only 50 features were expressed by all strains. Furthermore, direct profiling of isolated OMP using MALDI-TOF MS resulted in a characteristic spectral fingerprint for each 16S rRNA type. Fingerprints remained identical when intact cells instead of isolated OMP were analyzed. This finding suggests that the source of desorbed ions is the outer membrane. Based on the fingerprint we were able to assign 18 well-characterized clinical M. catarrhalis isolates to the correct subpopulation. Therefore, MALDI-TOF of intact M. catarrhalis provides a rapid and robust tool for M. catarrhalis strain typing that could be applied in epidemiological studies.     

Characterization of the Moraxella catarrhalis opa-like protein, OlpA, reveals a phylogenetically conserved family of outer membrane proteins

Moraxella catarrhalis is a human-restricted pathogen that can cause respiratory tract infections. In this study, we identified a previously uncharacterized 24-kDa outer membrane protein with a high degree of similarity to Neisseria Opa protein adhesins, with a predicted beta-barrel structure consisting of eight antiparallel beta-sheets with four surface-exposed loops. In striking contrast to the antigenically variable Opa proteins, the M. catarrhalis Opa-like protein (OlpA) is highly conserved and constitutively expressed, with 25 of 27 strains corresponding to a single variant. Protease treatment of intact bacteria and isolation of outer membrane vesicles confirm that the protein is surface exposed yet does not bind host cellular receptors recognized by neisserial Opa proteins. Genome-based analyses indicate that OlpA and Opa derive from a conserved family of proteins shared by a broad array of gram-negative bacteria.     

Purification of integral outer-membrane protein OmpC, a surface antigen from Salmonella typhi for structure-function studies: a method applicable to enterobacterial major outer-membrane protein

Extraction of the outer-membrane porin, OmpC, from Salmonella typhi Ty21a was done by using a modified salt-extraction procedure. It was possible to extract only the major outer-membrane protein (OMP) from the crude membrane using this method. Aberrant lipopolysaccharide (LPS) production in the galE mutant Ty21a has resulted in more isoforms of OmpC and subsequently led to anomalous mobility in SDS-PAGE. The purity of the preparation was confirmed by denaturing urea SDS-PAGE and N-terminal sequencing. The major OMP extracts had LPS of both bound and free forms. The free form of LPS could be removed by gel filtration and the bound form, largely, was removed using ion-exchange chromatography and by passing through ultrafiltration devices. This method has been used to extract the native trimer of OmpC, the major OMP, in a large scale, for structure-function studies. S. typhi Ty21a OmpC preparation yielded reproducible diffraction-quality crystals. Extracts of porin from wild-type Escherichia coli HB101, grown under high osmolarity conditions, showed a single species of OMP on SDS-PAGE. This suggests the possible application of the method to other gram-negative bacterial porins.     

Moraxella catarrhalis M35 is a general porin that is important for growth under nutrient-limiting conditions and in the nasopharynges of mice

Moraxella catarrhalis is a gram-negative respiratory pathogen that is an important causative agent for otitis media and exacerbations of chronic obstructive pulmonary disease. We have previously predicted the outer membrane protein M35 to be a general porin, and in the current study, we have investigated the function of M35 and its importance for survival of M. catarrhalis in vivo. Lipid bilayer experiments reveal that refolded M35 functions as a channel that is typical of gram-negative bacterial porins. M35 forms wide and water-filled channels with a single-channel conductance of about 1.25 nS in 1 M KCl solution and has only a small selectivity for cations over anions. When the in vitro growth characteristics of two M35 deletion mutant strains of M. catarrhalis were compared to the wild-type parent isolates, the growth of the mutant strains was inhibited only under nutrient-poor conditions. This growth defect could be eliminated by additional glutamic acid, but not additional aspartic acid, glycine, sucrose, or glucose. The mutant strains compensated for the lack of M35 by enhancing their uptake of glutamic acid, and this enhanced rate of glutamic acid uptake was attributed to the compensatory upregulation of a protein of approximately 40 kDa. M35 was also found to be essential for nasal colonization of mice, demonstrating that its presence is essential for survival of M. catarrhalis in vivo. These results suggest that M35 is a general porin that is necessary for the uptake of important energy sources by M. catarrhalis and that it is likely that M35 is an essential functional protein for in vivo colonization.     

The outer membrane protein of enteropathogenic Escherichia coli, described as the ''localised adherence factor'', is OmpF and probably not involved in adhesion to HEp-2 cells

Strains of enteropathogenic Escherichia coli (EPEC) were examined for a factor, described as an outer membrane protein (OMP) of 32 kilodaltons (kDa) and reported to be involved in the adhesion of EPEC to HeLa cells. A comparable OMP of 35 kDa was detected in strains of EPEC, although expression of this protein was not related to the ability of strains to adhere to HEp-2 cells. The 35 kDa OMP was found to be heat-modifiable and peptidoglycan associated, and considered to be the porin protein OmpF.     

Investigating the potential of conserved inner core oligosaccharide regions of Moraxella catarrhalis lipopolysaccharide as vaccine antigens: accessibility and functional activity of monoclonal antibodies and glycoconjugate derived sera

We investigated the conservation and antibody accessibility of inner core epitopes of Moraxella catarrhalis lipopolysaccharide (LPS) in order to assess their potential as vaccine candidates. Two LPS mutants, a single mutant designated lgt2 and a double mutant termed lgt2/lgt4, elaborating truncated inner core structures were generated in order to preclude expression of host-like outer core structures and to create an inner core structure that was shared by all three serotypes A, B and C of M. catarrhalis. Murine monoclonal antibodies (mAbs), designated MC2-1 and MC2-10 were obtained by immunising mice with the lgt2 mutant of M. catarrhalis serotype A strain. We showed that mAb MC2-1 can bind to the core LPS of wild-type (wt) serotype A, B and C organisms and concluded that mAb MC2-1 defines an immunogenic inner core epitope of M. catarrhalis LPS. We were unsuccessful in obtaining mAbs to the lgt2/lgt4 mutant. MAb MC2-10 only recognised the lgt2 mutant and the wt serotype A strain, and exhibited a strong requirement for the terminal N-acetyl-glucosamine residue of the lgt2 mutant core oligosaccharide, suggesting that this residue was immunodominant. Subsequently, we showed that both mAbs MC2-1 and MC2-10 could facilitate bactericidal killing of the lgt2 mutant, however neither mAb could facilitate bactericidal killing of the wt serotype A strain. We then confirmed and extended the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. catarrhalis wt strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes three conjugation strategies that either uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule, or a strong base treatment to remove all fatty acids from the LPS, thus creating amino functionalities in the lipid A region to conjugate via maleimide-thiol linker strategies targeting the carboxyl residues of the carrier protein and the free amino functionalities of the derived lipid A region of the carbohydrate resulted in a high loading of carbohydrates per carrier protein from these carbohydrate preparations. Immunisation derived antisera from rabbits recognised fully extended M. catarrhalis LPS and whole cells. Moreover, bactericidal activity was demonstrated to both the immunising carbohydrate antigen and importantly to wt cells, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by M. catarrhalis.     

Polymorphism of the major surface epitope of the CopB outer membrane protein of Moraxella catarrhalis

The CopB outer membrane protein has been considered a vaccine candidate for the prevention of infections due to Moraxella catarrhalis. Monoclonal antibody 10F3 recognizes whole cells of about 70% of clinical isolates, suggesting that this epitope is reasonably conserved. To determine whether CopB has other surface epitopes, we analyzed M. catarrhalis isolates using polyclonal sera against recombinant CopB proteins from a 10F3 positive isolate and a 10F3 negative isolate, and polyclonal sera against synthetic peptides that contained the sequence corresponding to the 10F3 epitope region of three different isolates. Extensive cross-reactivity was observed with the anti-CopB sera towards purified recombinant CopB proteins in Western blot and antigen ELISA, implying that antigenic regions common to both proteins were present. However, anti-CopB sera resembled anti-CopB peptide sera in exhibiting similar binding specificity to whole cells, segregating M. catarrhalis isolates into four CopB groups. We subsequently cloned and sequenced the copB genes from representative isolates. The deduced CopB amino acid sequences and the degree of sequence identity also demonstrated the existence of the same four CopB groups. Each of the four groups had a unique sequence in the 10F3 epitope region and a fifth group had the epitope deleted. The polymorphism of the major surface epitope prompts further consideration regarding the utility of CopB as a vaccine component as well as the design of an efficacious CopB-based vaccine to achieve broad protection against Moraxella infection.     

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.     

Conserved lipoprotein H.8 of pathogenic Neisseria consists entirely of pentapeptide repeats

The pathogenic Neisseria, N. gonorrhoeae and N. meningitidis, possess an outer membrane protein (OMP), designated H.8, with a conserved monoclonal antibody (MAb)-binding epitope. We determined the DNA sequence of a gonococcal H.8 gene, and confirmed the relationship between the cloned gene and the H.8 OMP by constructing a gonococcal mutant lacking H.8. The predicted H.8 OMP is a lipoprotein 71 amino acids in length, composed of 13 repeats of a consensus sequence AAEAP with perfect 5-residue periodicity. The AAEAP units form a repeating epitope that comprises the entire predicted sequence of the protein.     

The Moraxella catarrhalis outer membrane protein CD contains two distinct domains specifying adherence to human lung cells

Most Moraxella catarrhalis isolates express a highly-conserved outer membrane protein of 453 residues designated OMPCD, which has been previously shown to mediate binding to A549 human lung cells. Here, it is reported that two distinct domains of the M. catarrhalis strain O35E OMPCD protein specify adherence. Truncated proteins were expressed in Escherichia coli to demonstrate that OMPCD residues 1-240 as well as 241-400 are important for attachment to A549 cells, and database searches indicated that amino acids 285-299 resemble an adhesive motif found in eukaryotic proteins termed thrombospondin-type 3 repeat (TT3R). Cellular enzyme-linked immunosorbent assay using His-tagged proteins demonstrated that residues 236-300 of OMPCD, containing the TT3R motif, specify adhesive properties. Furthermore, these assays revealed that a purified protein encompassing residues 16-236 binds to A549 cells. The two cell-binding domains of OMPCD were further defined to amino acids 16-150 and 261-300 by utilizing a surface-display system, which was constructed from the M. catarrhalis autotransporter protein McaP, to express foreign peptides on the surface of recombinant bacteria.     

Class-3 porin protein of Neisseria meningitidis: cloning and structure of the gene.

The gene coding for the class-3 protein of Neisseria meningitidis was cloned and sequenced. The deduced amino acid (aa) sequence was highly homologous (50-78%) to those of other neisserial porin proteins. Alignment of the aa sequence of five neisserial porin proteins pinpointed several regions of identity or near identity. These are assumed to be membrane-spanning beta-strands. A comparison of the homologies between these neisserial porins showed that the class-3 protein is most closely related to the Neisseria gonorrhoeae P1A protein.     

Cloning and nucleotide sequence of the gene coding for the major 25-kilodalton outer membrane protein of Brucella abortus.

The cloning and sequencing of the Brucella abortus major 25-kDa outer membrane protein (OMP) is reported. The 25-kDa (group 3) OMP has been proposed, on the basis of amino acid composition, to be the counterpart of OmpA (D. R. Verstraete, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982). However, the amino acid sequence predicted from the cloned B. abortus gene did not reveal significant homology with either OmpA sequences from different members of the family Enterobacteriaceae or other known protein sequences.     

Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 microg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.     

鼠伤寒杆菌外膜蛋白作为保护性疫苗的初步实验研究

本文采用超声波破碎和Triton X-100处理、超速离心的方法,提取了鼠伤寒杆菌的外膜蛋白(OMP)。用提取的OMP免疫家兔和小鼠,经用ELISA方法测定小鼠、家兔及鼠伤寒意染患者血清中抗-OMP抗体含量,并用小鼠作足垫肿胀实验及主动和血清被动保护试验。结果免疫动物及患者血清中都含有较高滴度抗体,免疫小鼠足垫出现明显地DTH反应。50ug OMP免疫小鼠可保护500 LD50毒株的攻击,0.2ml免疫血清亦能够被动保护以上同样的毒株攻击。这些结果表明,提取的OMP有较强的免疫原性和明显的免疫保护作用,应用Western blot分析免疫血清均能识别36KD蛋白带,36KD蛋白带可能是鼠伤寒杆菌的主要免疫原。     

A Major Outer Membrane Protein of Rahnella aquatilis Functions as a Porin and Root Adhesin

A 38-kDa major outer membrane protein (OMP) was isolated from the nitrogen-fixing enterobacterium Rahnella aquatilis CF3. This protein exists as a stable trimer in the presence of 2% sodium dodecyl sulfate at temperatures below 60°C. Single channel experiments showed that this major OMP of R. aquatilis CF3 is able to form pores in the planar lipid membrane. Two oligonucleotides encoding the N-terminal portion of the 38-kDa OMP and C-terminal portion of OmpC were used to amplify the 38-kDa gene by PCR. The deduced amino acid sequence showed a strong homology with Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, and Serratia marcescens OmpC sequences, except loops L6 and L7, which are postulated to be cell surface exposed. On the basis of the OmpF-PhoE three-dimensional structure, it seems likely that this 38-kDa organizes three 16-strand β-barrel subunits. The relationship between the structure and the double functionality of this protein as porin and as a root adhesin is discussed.     

Relationship of the human immunodeficiency virus type 1 gp120 third variable loop to a component of the CD4 binding site in the fourth conserved region.

Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions.     

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.