scholarly journals Immunogenicity of Mannheimia haemolytica Recombinant Outer Membrane Proteins Serotype 1-Specific Antigen, OmpA, OmpP2, and OmpD15

2011 ◽  
Vol 18 (12) ◽  
pp. 2067-2074 ◽  
Author(s):  
Sahlu Ayalew ◽  
Binu Shrestha ◽  
Marie Montelongo ◽  
Amanda E. Wilson ◽  
Anthony W. Confer
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Recombinant Proteins ◽  
Outer Membrane Proteins ◽  
Specific Antigen ◽  
Antibody Responses ◽  
Mannheimia Haemolytica ◽  
Content Type ◽  
Outer Membranes ◽  
Serotype 1

ABSTRACTWe previously identifiedMannheimia haemolyticaouter membrane proteins (OMPs) that may be important immunogens by using immunoproteomic analyses. Genes for serotype 1-specific antigen (SSA-1), OmpA, OmpP2, and OmpD15 were cloned and expressed, and recombinant proteins were purified. Objective 1 of this study was to demonstrate immunogenicity of the four recombinant OMPs in mice and cattle. Objective 2 was to determine if the addition of individual recombinant OMPs or combinations of them would modify immune responsiveness of mice to the recombinant chimeric protein SAC89, containing the main epitope fromM. haemolyticaouter membrane lipoprotein PlpE and the neutralizing epitope ofM. haemolyticaleukotoxin. Mice vaccinated with recombinant OmpA (rOmpA), rSSA-1, rOmpD15, and rOmpP2 developed significant antibody responses toM. haemolyticaouter membranes and to the homologous recombinant OMP. Cattle vaccinated with rOmpA and rSSA-1 developed significant antibodies toM. haemolyticaouter membranes by day 28, whereas cattle vaccinated with rOmpD15 and rOmpP2 developed only minimal responses. Sera from cattle vaccinated with each of the recombinant proteins stimulated complement-mediated killing of the bacterium. Concurrent vaccination with SAC89 plus any of the four rOMPs singly resulted in increased endpoint anti-SAC89 titers, and for the SAC89/rSSA-1 vaccinees, the response was increased significantly. In contrast, the SAC89/P2/SSA-1 and SAC89/OmpA/P2/D15/SSA-1 combination vaccines resulted in significant decreases in anti-SAC89 antibodies compared to SAC89 vaccination alone. In conclusion, under the conditions of these experiments, vaccination of mice and cattle with rOmpA and rSSA-1 stimulated high antibody responses and may have protective vaccine potential.

scholarly journals A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria

2016 ◽  
Vol 291 (32) ◽  
pp. 16720-16729 ◽  
Author(s):  
Yan Wang ◽  
Rui Wang ◽  
Feng Jin ◽  
Yang Liu ◽  
Jiayu Yu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Outer Membranes

scholarly journals A Disulfide Oxidoreductase (CHU_1165) Is Essential for Cellulose Degradation by Affecting Outer Membrane Proteins in Cytophaga hutchinsonii

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Dong Zhao ◽  
Ying Wang ◽  
Sen Wang ◽  
Weican Zhang ◽  
Qingsheng Qi ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Disulfide Bond ◽  
Cellulose Degradation ◽  
Outer Membrane Proteins ◽  
Hypothetical Protein ◽  
Pleiotropic Effect ◽  
Content Type ◽  
Cytophaga Hutchinsonii ◽  
Disulfide Oxidoreductase

ABSTRACT Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii. Further study showed that cells of the Δ1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro. Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins. IMPORTANCE Cytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii. Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.

scholarly journals Catecholamine-Modulated Novel Surface-Exposed Adhesin LIC20035 ofLeptospiraspp. Binds Host Extracellular Matrix Components and Is Recognized by the Host during Infection

2017 ◽  
Vol 84 (6) ◽  
Author(s):  
Karukriti Kaushik Ghosh ◽  
Aman Prakash ◽  
Vinayagamurthy Balamurugan ◽  
Manish Kumar
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Membrane Surface ◽  
Outer Membrane Proteins ◽  
Extracellular Matrices ◽  
Content Type ◽  
Extracellular Matrix Components ◽  
Gene Transcripts ◽  
Host Tissues

ABSTRACTIn this study, the effect of the host stress hormone catecholamine onLeptospiragene transcripts encoding outer membrane proteins was investigated. There was no impact of catecholamine supplementation on thein vitrogrowth pattern ofLeptospira interrogans; however, 7 genes out of 41 were differentially transcribed, and the effect was reversed to the basal level in the presence of the antagonist propranolol. Comprehensive analysis of one of the differentially regulated proteins, LIC20035 (in serovar Copenhageni)/LB047 (in serovar Lai) (due to catecholamine supplementation), revealed immunogenicity and ability to adhere to host extracellular matrices. Protease accessibility assay and phase partition of integral membrane proteins ofLeptospirashowed LIC20035/LB047 to be an outer membrane surface-exposed protein. The recombinant LIC20035 protein can be serologically detected using human/bovine sera positive for leptospirosis. Moreover, the recombinant LIC20035 can bind to diverse host extracellular matrices, with a higher affinity toward collagen and chondroitin sulfate.IMPORTANCELeptospirosis is a neglected tropical disease of global importance. This study aimed to identify outer membrane proteins of pathogenicLeptospiraresponding to host chemical signals like catecholamines, with the potential to serve as virulence factors, new serodiagnostic antigens, and vaccine candidates. This study mimicked the plausible means by whichLeptospiraduring infection and hormonal stress intercepts host catecholamines to disseminate in host tissues.

scholarly journals Putative β-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
G. J. Staton ◽  
S. D. Carter ◽  
S. Ainsworth ◽  
J. Mullin ◽  
R. F. Smith ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Functional Characterization ◽  
Signal Peptidase ◽  
Effective Vaccine ◽  
Digital Dermatitis ◽  
Diverse Range ◽  
Content Type ◽  
Signal Peptidase I

ABSTRACT Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

scholarly journals Immunization with Ehrlichia P28 Outer Membrane Proteins Confers Protection in a Mouse Model of Ehrlichiosis

2011 ◽  
Vol 18 (12) ◽  
pp. 2018-2025 ◽  
Author(s):  
Patricia A. Crocquet-Valdes ◽  
Nagaraja R. Thirumalapura ◽  
Nahed Ismail ◽  
Xuejie Yu ◽  
Tais B. Saito ◽  
...  
Keyword(s):  
T Cells ◽  
Membrane Proteins ◽  
Immune Responses ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
The United States ◽  
Mononuclear Phagocytes ◽  
Specific Cell ◽  
Content Type ◽  
Ifn Γ

ABSTRACTThe obligately intracellular bacteriumEhrlichia chaffeensisthat resides in mononuclear phagocytes is the etiologic agent of human monocytotropic ehrlichiosis (HME). HME is an emerging and often life-threatening, tick-transmitted infectious disease in the United States. Effective primary immune responses againstEhrlichiainfection involve generation ofEhrlichia-specific gamma interferon (IFN-γ)-producing CD4+T cells and cytotoxic CD8+T cells, activation of macrophages by IFN-γ, and production ofEhrlichia-specific antibodies of the Th1 isotype. Currently, there are no vaccines available against HME. We evaluated the ability of 28-kDa outer membrane proteins (P28-OMP-1) of the closely relatedEhrlichia muristo stimulate long-term protective memory T and B cell responses and confer protection in mice. The spleens of mice vaccinated withE. murisP28-9, P28-12, P28-19, or a mixture of these three P28 proteins (P28s) using a DNA prime-protein boost regimen and challenged withE. murishad significantly lower bacterial loads than the spleens of mock-vaccinated mice. Mice immunized with P28-9, P28-12, P28-19, or the mixture inducedEhrlichia-specific CD4+Th1 cells. Interestingly, mice immunized with P28-14, orthologs of which inE. chaffeensisandE. canisare primarily expressed in tick cells, failed to lower the ehrlichial burden in the spleen. Immunization with the recombinant P28-19 protein alone also significantly decreased the bacterial load in the spleen and liver compared to those of the controls. Our study reports, for the first time, the protective roles of theEhrlichiaP28-9 and P28-12 proteins in addition to confirming previous reports of the protective ability of P28-19. Partial protection induced by immunization with P28-9, P28-12, and P28-19 againstEhrlichiawas associated with the generation ofEhrlichia-specific cell-mediated and humoral immune responses.

scholarly journals DEVELOPMENT OF THE VACCINE BASED ON THE RECOMBINANT ANTIGENS OF PSEUDOMONAS AERUGINOSA

2019 ◽  
Vol 1 (1) ◽  
pp. 74-80
Author(s):  
N. A. Mihailova ◽  
E. M. Zimina ◽  
A. V. Soldatenkova ◽  
A. A. Kaloshin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Proteins ◽  
Fusion Protein ◽  
Outer Membrane ◽  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Outer Membrane Proteins ◽  
Recombinant Fusion Protein ◽  
Recombinant Antigens ◽  
Recombinant Fusion Proteins

Aim. The aim is obtaining, investigation and selection of recombinant antigens for inclusion theirs into the against Pseudomonas vaccine. Materials and methods. The genes encoding of the outer  membrane proteins F, L and I and Exotoxin A were synthesized by PCR with the genomic DNA of Pseudomonas aeruginosa. The amplified sequences were cloned into plasmid vectors for expression in cells of Escherichia coli. As the result of expression were the synthesized recombinant proteins that were purified in columns with a nickel-activated sorbent. The authenticity of the recombinant antigens was assessed by electrophoresis and immunoblotting. For assessing the immunogenicity of the recombinant proteins,they were sorbed on aluminum hydroxide and used for intraperitoneal immunization of mice. After a course of immunization, mice were injected intraperitoneally with a live virulent culture or еxotoxin A. Results. The obtained recombinant outer membrane proteins OprF, OprL and OprI, as well as the deletion variant of еxotoxin A (toxoid) stimulated immune reactions and protected the experimental animals from the virulent culture of P. aeruginosa. Using of the complexes of the recombinant proteins, as well as immunization with the fusion proteins consisting from sequences of two or three recombinant antigens, produced an additive increase in protective effects. The combination of the recombinant OprF protein and the recombinant toxoid (efficiency index of protective properties (EI 3.0) and two recombinant fusion proteins (EI 3.5) were the most effective. The first recombinant fusion protein (OprF-aTox-OprI) consisted from fused polypeptide sequences of OprF, toxoid and OprI. The second recombinant fusion protein (OprF-OprI) consisted from fused polypeptide sequences of OprF and OprI. Conclusion. The data obtained showed the fundamental possibility of using recombinant fusion proteins OprF-aTox-OprI and OprF-OprI as well as the complex of the recombinant OprF protein and the recombinant toxoid as the candidated vaccines against Pseudomonas aeruginosa.

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