scholarly journals Fc-Dependent Polyclonal Antibodies and Antibodies to Outer Membrane Proteins A and B, but Not to Lipopolysaccharide, Protect SCID Mice against Fatal Rickettsia conorii Infection

2004 ◽  
Vol 72 (4) ◽  
pp. 2222-2228 ◽  
Author(s):  
Hui-Min Feng ◽  
Ted Whitworth ◽  
Juan P. Olano ◽  
Vsevolod L. Popov ◽  
David H. Walker
Keyword(s):  
Monoclonal Antibodies ◽  
Outer Membrane ◽  
Spotted Fever ◽  
Outer Membrane Proteins ◽  
Severe Combined Immunodeficiency ◽  
Scid Mice ◽  
Rickettsia Conorii ◽  
Antibody Treatment ◽  
Fab Fragments

ABSTRACT An emphasis on cellular immunity against Rickettsia has led to neglect of analysis of the role of antibody. The availability of an excellent mouse model of spotted fever rickettsiosis enabled investigation of a potential role of antibody in immunity to Rickettsia conorii. C3H severe combined immunodeficiency (SCID) mice were passively transfused with monoclonal antibodies against rickettsial outer membrane protein A (OmpA), OmpB, or lipopolysaccharide (LPS), polyclonal anti-R. conorii serum, Fab fragments of polyclonal antiserum, or no antibodies and then challenged 48 h later with 10 50% lethal doses (LD50) of R. conorii. All mice that received monoclonal antibodies against OmpA and two of four mice that received monoclonal antibodies against OmpB or polyclonal antisera were completely protected, but the recipients of anti-LPS antibodies or the Fab fragments were not protected. Polyclonal antibody treatment of C3H SCID mice that had been infected with 10 LD50 of R. conorii 4 or 5 days earlier prolonged the life of the infected mice from 10.4 to 22.5 days and resulted in decreased levels of infectious rickettsiae in the spleen and liver 24 and 48 h later. Treatment with protective antibodies resulted in the development of large aggregates of R. conorii antigens in splenic macrophages and intraphagolysosomal rickettsial death and digestion. The kinetics of development of antibodies to R. conorii determined by immunoblotting revealed antibodies to LPS on day 6 and antibodies to OmpA and OmpB on day 12, when recovery from the infection had already occurred. Antibodies to particular epitopes of OmpA and OmpB may protect against reinfection, but they may not play a key role in immunity against primary infection. Antibodies might be useful for treating infections with antibiotic-resistant organisms, and some B-cell epitopes should be included in a subunit vaccine.

scholarly journals Role of Phosphatidylethanolamine in the Biogenesis of Mitochondrial Outer Membrane Proteins

2013 ◽  
Vol 288 (23) ◽  
pp. 16451-16459 ◽  
Author(s):  
Thomas Becker ◽  
Susanne E. Horvath ◽  
Lena Böttinger ◽  
Natalia Gebert ◽  
Günther Daum ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Proper Function ◽  
Mitochondrial Outer Membrane ◽  
Tom Complex ◽  
Precursor Proteins ◽  
Helical Proteins ◽  
The Stability

The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.

scholarly journals The role of BamA in the biogenesis of beta-barrel membrane proteins

2014 ◽  
Vol 70 (a1) ◽  
pp. C578-C578
Author(s):  
Nicholas Noinaj ◽  
Adam Kuszak ◽  
Curtis Balusek ◽  
JC Gumbart ◽  
Petra Lukacik ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Crystal Structures ◽  
Outer Membrane Proteins ◽  
Hydrophobic Surface ◽  
Md Simulations ◽  
Structural Features ◽  
Bam Complex ◽  
Beta Barrel

Beta-barrel membrane proteins are essential for nutrient import, signaling, motility, and survival. In Gram-negative bacteria, the beta-barrel assembly machinery (BAM) complex is responsible for the biogenesis of beta-barrel outer membrane proteins (OMPs), with homologous complexes found in mitochondria and chloroplasts. Despite their essential roles, exactly how these OMPs are formed remains unknown. The BAM complex consists of a central and essential component called BamA (an OMP itself) and four lipoproteins called BamB-E. While the structure of the lipoproteins have been reported, the structure of full length BamA has been elusive. Recently though, we described the structure of BamA from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane beta-barrel domain. Together, our crystal structures and molecule dynamics (MD) simulations revealed several structural features which gave clues to the mechanism by which BamA catalyzes beta-barrel assembly. The first is that the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the beta-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. Third, the beta-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane. And fourth, a surface exposed exit pore positioned above the lateral opening site which may play a role in the biogenesis of extracellular loops. In this presentation, the crystal structures and MD simulations of BamA will be presented along with our work looking at the role of these four structural features in the role of BamA within the BAM complex.

scholarly journals Two Outer Membrane Proteins Are Required for Maximal Type I Secretion of the Caulobacter crescentus S-Layer Protein

2004 ◽  
Vol 186 (23) ◽  
pp. 8000-8009 ◽  
Author(s):  
Michael C. Toporowski ◽  
John F. Nomellini ◽  
Peter Awram ◽  
John Smit
Keyword(s):  
Outer Membrane ◽  
Caulobacter Crescentus ◽  
Outer Membrane Proteins ◽  
Functional System ◽  
Type I ◽  
Wild Type ◽  
Residual Level ◽  
The Individual ◽  
Multicopy Vector

ABSTRACT Transport of RsaA, the crystalline S-layer subunit protein of Caulobacter crescentus, is mediated by a type I secretion mechanism. Two proteins have been identified that play the role of the outer membrane protein (OMP) component in the RsaA secretion machinery. The genes rsaF a and rsaF b were identified by similarity to the Escherichia coli hemolysin secretion OMP TolC by using the C. crescentus genome sequence. The rsaF a gene is located several kilobases downstream of the other transporter genes, while rsaF b is completely unlinked. An rsaF a knockout had ∼56% secretion compared to wild-type levels, while the rsaF b knockout reduced secretion levels to ∼79%. When expression of both proteins was eliminated, there was no RsaA secretion, but a residual level of ∼9% remained inside the cell, suggesting posttranslational autoregulation. Complementation with either of the individual rsaF genes by use of a multicopy vector, which resulted in 8- to 10-fold overexpression of the proteins, did not restore RsaA secretion to wild-type levels, indicating that both rsaF genes were required for full-level secretion. However, overexpression of rsaFa (with normal rsaF b levels) in concert with overexpression of rsaA resulted in a 28% increase in RsaA secretion, indicating a potential for significantly increasing expression levels of an already highly expressing type I secretion system. This is the only known example of type I secretion requiring two OMPs to assemble a fully functional system.

scholarly journals Purification of the Major Outer Membrane Protein of Azospirillum brasilense, Its Affinity to Plant Roots, and Its Involvement in Cell Aggregation

2001 ◽  
Vol 14 (4) ◽  
pp. 555-561 ◽  
Author(s):  
Saul Burdman ◽  
Gabriella Dulguerova ◽  
Yaacov Okon ◽  
Edouard Jurkevitch
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Azospirillum Brasilense ◽  
Outer Membrane Proteins ◽  
Cell Aggregation ◽  
Major Outer Membrane Protein ◽  
Adhesion Assay ◽  
Fab Fragments

The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

scholarly journals Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

2012 ◽  
Vol 23 (20) ◽  
pp. 3948-3956 ◽  
Author(s):  
Maria Bohnert ◽  
Lena-Sophie Wenz ◽  
Ralf M. Zerbes ◽  
Susanne E. Horvath ◽  
David A. Stroud ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Early Stage ◽  
Outer Membrane Proteins ◽  
Mitochondrial Outer Membrane ◽  
Large Core ◽  
Large Protein ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Protein Biogenesis

Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins.

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