scholarly journals Regulated Delayed Expression of rfaH in an Attenuated Salmonellaenterica Serovar Typhimurium Vaccine Enhances Immunogenicity of Outer Membrane Proteins and a Heterologous Antigen

2009 ◽  
Vol 77 (12) ◽  
pp. 5572-5582 ◽  
Author(s):  
Qingke Kong ◽  
Qing Liu ◽  
Kenneth L. Roland ◽  
Roy Curtiss
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Lethal Dose ◽  
Enteric Bacteria ◽  
Lymphoid Tissues ◽  
Heterologous Antigen ◽  
O Antigen ◽  
Mutant Strains ◽  
Serovar Typhimurium

ABSTRACT RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A ΔrfaH mutant strain is attenuated in mice (50% lethal dose [LD50], >108 CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC PBAD promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the PBAD promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, ΔPrfaH178, was introduced into the attenuated vaccine strain χ9241 (ΔpabA ΔpabB ΔasdA) expressing the pneumococcal surface protein PspA from an Asd+ balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic ΔrfaH derivative or the isogenic RfaH+ parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD50 of virulent S treptococcus pneumoniae WU2. Immunization with ΔPrfaH178 mutant strains led to increased levels of protection compared to that of the parent χ9241 and of a ΔrfaH derivative of χ9241.

scholarly journals Immunization withSalmonella entericaSerovar Typhimurium-Derived Outer Membrane Vesicles Delivering the Pneumococcal Protein PspA Confers Protection against Challenge withStreptococcus pneumoniae

2010 ◽  
Vol 79 (2) ◽  
pp. 887-894 ◽  
Author(s):  
Maneesha Muralinath ◽  
Meta J. Kuehn ◽  
Kenneth L. Roland ◽  
Roy Curtiss
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Lethal Dose ◽  
Serum Antibody ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Gram Negative Bacteria ◽  
Vesicle Preparation ◽  
Serovar Typhimurium

ABSTRACTGram-negative bacteria produce outer membrane vesicles (OMVs) that serve a variety of functions related to survival and pathogenicity. Periplasmic and outer membrane proteins are naturally captured during vesicle formation. This property has been exploited as a method to derive immunogenic vesicle preparations for use as vaccines. In this work, we constructed aSalmonella entericaserovar Typhimurium strain that synthesized a derivative of the pneumococcal protein PspA engineered to be secreted into the periplasmic space. Vesicles isolated from this strain contained PspA in the lumen. Mice intranasally immunized with the vesicle preparation developed serum antibody responses against vesicle components that included PspA andSalmonella-derived lipopolysaccharide and outer membrane proteins, while no detectable responses developed in mice immunized with an equivalent dose of purified PspA. Mucosal IgA responses developed against theSalmonellacomponents, while the response to PspA was less apparent in most mice. Mice immunized with the vesicle preparation were completely protected against a 10× 50% lethal dose (LD50) challenge ofStreptococcus pneumoniaeand significantly protected against a 200× LD50challenge, while control mice immunized with purified PspA or empty vesicles were not protected. These results establish that vesicles can be used to mucosally deliver an antigen from a Gram-positive organism and induce a protective immune response.

scholarly journals Immune Responses and Protective Immunity in Pangasius Pangasius (Hamilton, 1822) as Induced by Outer Membrane Proteins of Edwardsiella Tarda and Aluminium Hydroxide Adjuvant Complex

2022 ◽  
Author(s):  
Harresh Adikesavalu ◽  
Thangapalam Jawahar Abraham ◽  
Siddhartha Narayan Joardar
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Antibody Production ◽  
Aluminium Hydroxide ◽  
Lymphocyte Proliferation ◽  
Protective Immunity ◽  
Outer Membrane Proteins ◽  
Lethal Dose ◽  
Serum Antibody ◽  
Edwardsiella Tarda

Abstract Edwardsiella tarda is considered one of the important bacterial fish pathogens. The outer membrane proteins (OMPs) of E. tarda are structurally and functionally conserved, and immunogenic. This study assessed the effects of the OMPs of E. tarda CGH9 as a vaccine without aluminium hydroxide [AH] (T1) and with AH adjuvant (T2) on the respiratory burst (ROB) activity, lymphocyte proliferation of head kidney (HK) leukocytes, and serum antibody production in pangas catfish Pangasius pangasius. The ROB activity and lymphocyte proliferation of HK leukocytes increased in both vaccinated groups compared to control. Nonetheless, the T2 group showed a gradual increase in ROB activity and lymphocyte proliferation of HK leukocytes up to 3-weeks post-vaccination (wpv). The serum antibody production in the T1 group decreased initially for up to 2-wpv and increased from 3-wpv; whereas, in the T2 group, the serum-specific antibody levels were significantly high from 1-wpv compared to control. Simultaneously, the protective efficacy in terms of relative percentage survival (RPS) in the T2 group after injecting with a lethal dose of E. tarda CGH9 was high (89.00±15.56) compared to the T1 group (78.00±0.00). Furthermore, the catfish administered with a booster dose of E. tarda OMPs with or without AH adjuvant showed no additional increase in immune response or protective immunity. These results suggested that E. tarda OMPs and AH adjuvant complex has a higher potential to induce protective immunity, which may be a good choice as a vaccine to combat E. tarda infection in catfish.

scholarly journals Outer Membrane Proteins of Fibrobacter succinogenes with Potential Roles in Adhesion to Cellulose and in Cellulose Digestion

2007 ◽  
Vol 189 (19) ◽  
pp. 6806-6815 ◽  
Author(s):  
Hyun-Sik Jun ◽  
Meng Qi ◽  
Joshua Gong ◽  
Emmanuel E. Egbosimba ◽  
Cecil W. Forsberg
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Binding Proteins ◽  
Outer Membrane Proteins ◽  
Mass Spectrometry Analysis ◽  
Crystalline Cellulose ◽  
Fibrobacter Succinogenes ◽  
Essential Components ◽  
Mutant Strains ◽  
Cellulose Binding

ABSTRACT Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. succinogenes.

scholarly journals Inactivation of ompX Causes Increased Interactions of Type 1 Fimbriated Escherichia coli with Abiotic Surfaces

2004 ◽  
Vol 186 (1) ◽  
pp. 226-234 ◽  
Author(s):  
Karen Otto ◽  
Malte Hermansson
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Phenotypic Characterization ◽  
Phenotypic Changes ◽  
Mutant Strains ◽  
Surface Contact

ABSTRACT During the initial steps of biofilm formation, bacteria have to adapt to a major change in their environment. The adhesion-induced phenotypic changes in a type 1 fimbriated Escherichia coli strain included reductions in the levels of several outer membrane proteins, one of which was identified as OmpX. Here, the phenotypes of mutant strains that differ at the ompX locus were studied with regard to adhesion, cell surface properties, and resistance to stress and antimicrobial compounds. The kinetics of adhesion were measured online by an extended quartz crystal microbalance technique for wild-type and mutant strains with a fimbriated or nonfimbriated background. Deletion of ompX led to significantly increased cell-surface contact in fimbriated strains but to decreased cell-surface contact in a nonfimbriated strain. Phenotypic characterization of the ompX mutant demonstrated that ompX interferes with proper regulation of cell surface structures that play a key role in mediating firm contact of the cell with a surface (i.e., type 1 fimbriae, flagellae, and exopolysaccharides). These phenotypic changes were accompanied by increased tolerance to several antibiotic compounds and sodium dodecyl sulfate. Based on these results, we propose that changes in the composition of outer membrane proteins during fimbria-mediated adhesion may be part of a coordinated adaptive response to the attached mode of growth.

scholarly journals Surface Antigen Exposure by Bismuth Dimercaprol Suppression of Klebsiella pneumoniae Capsular Polysaccharide

1999 ◽  
Vol 67 (2) ◽  
pp. 664-669 ◽  
Author(s):  
Philip Domenico ◽  
J. M. Tomas ◽  
S. Merino ◽  
X. Rubires ◽  
Burke A. Cunha
Keyword(s):  
Membrane Proteins ◽  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Capsular Polysaccharide ◽  
Outer Membrane Proteins ◽  
Defined Medium ◽  
Phagocytic Index ◽  
Dependent Manner ◽  
O Antigen ◽  
Bacterial Capsule

ABSTRACT The bacterial capsule is an important virulence determinant in animal and plant disease. Bacterial capsule and slime can be inhibited by bismuth compounds, especially when complexed with lipophilic thiol chelators. Bismuth dimercaprol (BisBAL) at 1 ppm of Bi3+repressed Klebsiella pneumoniae capsule expression in defined medium by nearly 90%, which exposed subsurface structures. The phagocytic index for BisBAL-treated bacteria increased from <10 to 360 bacteria per 100 neutrophils in the presence of complement and anticapsular or anti-O antigen antiserum. BisBAL treatment also enhanced the reactivity of monoclonal antibodies (MAbs) specific for the O1-antigen lipopolysaccharide (LPS) or the LPS core in a dose-dependent manner as indicated by the results of enzyme-linked immunosorbent assays. When anti-O1 MAb was used, the reactivity increased significantly for fully encapsulated O1:K1 or O1:K2 cells but not for O1:K− cells. Deposition of C3b also increased significantly for BisBAL-treated O1:K1 or O1:K2 cells but not for O1:K− cells. Survival of a serum-sensitive strain was <0.1% when nonimmune human serum absorbed with O1:K1 cells was used and 107% when BisBAL-treated cells were used for absorption. Outer membrane proteins were also more accessible on the surface of K. pneumoniae after BisBAL treatment. Thus, at subinhibitory levels, BisBAL inhibited capsule expression, which promoted phagocytosis, enhanced the reactivity of specific antibodies for LPS O antigen, LPS core epitopes, or outer-membrane proteins, and enhanced complement interaction with encapsulated K. pneumoniae. By unmasking bacterial surface structures and enhancing the immune system reactivity to bacteria, bismuth thiols may prove useful as adjuncts for vaccination.

scholarly journals Characterization of an Outer Membrane Protein of Salmonella enterica Serovar Typhimurium That Confers Protection against Typhoid

2008 ◽  
Vol 15 (9) ◽  
pp. 1461-1471 ◽  
Author(s):  
Nowsheen Hamid ◽  
S. K. Jain
Keyword(s):  
Molecular Mass ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Outer Membrane Proteins ◽  
Lethal Dose ◽  
Health Concern ◽  
Delayed Type Hypersensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Database ◽  
Serovar Typhimurium

ABSTRACT Typhoid caused by Salmonella enterica serovar Typhi remains a major health concern worldwide. The emergence of multidrug-resistant strains of Salmonella with increased virulence, communicability, and survivability leading to increased morbidity and mortality has further complicated its management. Currently available vaccines for typhoid have less-than-desired efficacy and certain unacceptable side effects, making it pertinent to search for new immunogens suitable for vaccine formulation. The outer membrane proteins (OMPs) of Salmonella have been considered possible candidates for conferring protection against typhoid. OMPs interface the cell with the environment, thus representing important virulence factors with a significant role in the pathobiology of gram-negative bacteria and bacterial adaptation. An OMP of Salmonella enterica serovar Typhimurium with an apparent molecular mass of 49 kDa that is highly immunogenic, evokes humoral and cell-mediated immune responses, and confers 100% protection to immunized rats against challenge with very high doses (up to 100 times the 50% lethal dose) of Salmonella enterica serovar Typhimurium has been identified. Further, very efficient clearance of bacteria from the reticuloendothelial systems of immunized animals was seen. This protein is recognized by the antibodies present in serum of typhoid patients. When sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel-eluted protein was further analyzed by high-performance liquid chromatography (HPLC) and two-dimensional electrophoresis, two polypeptides with the same molecular weight were resolved. These have different isoelectric points and gave two peaks with different retention times in reverse-phase HPLC. However, only one of the two bands interacted with patient serum. The immunogenicity studies (enzyme-linked immunosorbent assay and delayed-type hypersensitivity [DTH]) indicated that the immunoreactive protein evoked a strong immune response in rats. The N-terminal sequencing and analysis of the homology of this protein with sequences in the protein database of Salmonella resulted in a match with the N-terminal sequences of a protein in Salmonella enterica serovar Typhi (CT18 and Ty2 strains). The homology search further revealed it to be a hypothetical protein, whose gene had unidentified open reading frames in Salmonella serovar Typhi encoding 447 amino acid residues, corresponding to a molecular mass of 49 kDa. The nucleotide sequence of the encoding gene was deduced, and the gene was amplified by PCR using appropriate primers. An amplified 1.3-kb band was purified and sequenced to confirm its identity. These OMPs provide promising targets for the development of a candidate vaccine against typhoid.

scholarly journals Correlation between Ceftriaxone Resistance of Salmonella enterica Serovar Typhimurium and Expression of Outer Membrane Proteins OmpW and Ail/OmpX-Like Protein, Which Are Regulated by BaeR of a Two-Component System

2005 ◽  
Vol 49 (9) ◽  
pp. 3955-3958 ◽  
Author(s):  
Wensi S. Hu ◽  
Pei-Chuan Li ◽  
Chao-Yin Cheng
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Outer Membrane Proteins ◽  
Regulator Gene ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Serovar Typhimurium

ABSTRACT Mutant 7F2 of Salmonella enterica serovar Typhimurium has a transposon inserted in the regulator gene baeR of a two-component system and showed a more-than-fourfold reduction in resistance to ceftriaxone. Complementation analysis suggested an association among the outer membrane proteins OmpW and STM3031, ceftriaxone resistance, and baeR.

scholarly journals Enterobacterial Common Antigen Mutants of Salmonella enterica Serovar Typhimurium Establish a Persistent Infection and Provide Protection against Subsequent Lethal Challenge

2011 ◽  
Vol 80 (1) ◽  
pp. 441-450 ◽  
Author(s):  
Jeremy J. Gilbreath ◽  
Jennifer Colvocoresses Dodds ◽  
Paul D. Rick ◽  
Mark J. Soloski ◽  
D. Scott Merrell ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Enteric Bacteria ◽  
Rank Test ◽  
Common Antigen ◽  
Lethal Challenge ◽  
Heterologous Antigen ◽  
Content Type ◽  
Enterobacterial Common Antigen ◽  
Mutant Strains ◽  
Serovar Typhimurium

ABSTRACTInfection withSalmonellaspp. is a significant source of disease globally. A substantial proportion of these infections are caused bySalmonella entericaserovar Typhimurium. Here, we characterize the role of the enterobacterial common antigen (ECA), a surface glycolipid ubiquitous among enteric bacteria, inS.Typhimurium pathogenesis. Construction of a defined mutation in the UDP-N-acetylglucosamine-1-phosphate transferase gene,wecA, in two clinically relevant strains ofS.Typhimurium, TML and SL1344, resulted in strains that were unable to produce ECA. Loss of ECA did not affect the gross cell surface ultrastructure, production of lipopolysaccharide (LPS), flagella, or motility. However, thewecAmutant strains were attenuated in both oral and intraperitoneal mouse models of infection (P< 0.001 for both routes of infection; log rank test), and virulence could be restored by complementation of thewecAgene intrans. Despite the avirulence of the ECA-deficient strains, thewecAmutant strains were able to persistently colonize systemic sites (spleen and liver) at moderate levels for up to 70 days postinfection. Moreover, immunization with thewecAmutant strains provided protection against a subsequent lethal oral or intraperitoneal challenge with wild-typeS.Typhimurium. Thus,wecAmutant (ECA-negative) strains ofSalmonellamay be useful as live attenuated vaccine strains or as vehicles for heterologous antigen expression.

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