scholarly journals The Major Phase-Variable Outer Membrane Protein ofEscherichia coli Structurally Resembles the Immunoglobulin A1 Protease Class of Exported Protein and Is Regulated by a Novel Mechanism Involving Dam and OxyR

1999 ◽  
Vol 181 (7) ◽  
pp. 2132-2141 ◽  
Author(s):  
Ian R. Henderson ◽  
Peter Owen
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Dna Sequences ◽  
Outer Membrane Protein ◽  
Signal Sequence ◽  
Regulatory Region ◽  
Specific Antigen ◽  
Phase Variation ◽  
Phase Variable ◽  
Phase Switching

ABSTRACT Here we report the characterization of an Escherichia coli gene (agn43) which encodes the principal phase-variable outer membrane protein termed antigen 43 (Ag43). Theagn43 gene encodes a precursor protein of 107 kDa containing a 52-amino-acid signal sequence. Posttranslational processing generates an α43 subunit (predictedM r of 49,789) and a C-terminal domain (β43) with features typical of a bacterial integral outer membrane protein (predicted M r of 51,642). Secondary structure analysis predicts that β43 exists as an 18-stranded β barrel and that Ag43 shows structural organization closely resembling that of immunoglobulin A1 protease type of exoprotein produced by pathogenic Neisseria andHaemophilus spp. The correct processing of the polyprotein to α43 and β43 in OmpT, OmpP, and DegP protease-deficient E. coli strains points to an autocatalytic cleavage mechanism, a hypothesis supported by the occurrence of an aspartyl protease active site within α43. Ag43, a species-specific antigen, possesses two RGD motifs of the type implicated in binding to human integrins. The mechanism of reversible phase variation was studied by immunochemical analysis of a panel of well-defined regulatory mutants and by analysis of DNA sequences upstream of agn43. Evidence strongly suggests that phase variation is regulated by both deoxyadenosine methylase (Dam) and by OxyR. Thus, oxyR mutants are locked on for Ag43 expression, whereas dam mutants are locked off for Ag43 expression. We propose a novel mechanism for the regulation of phase switching in which OxyR competes with Dam for unmethylated GATC sites in the regulatory region of the agn43 gene.

scholarly journals Transcriptional and immunological analysis of the putative outer membrane protein and vaccine candidate TprL of Treponema pallidum

2020 ◽  
Author(s):  
Austin M. Haynes ◽  
Mark Fernandez ◽  
Emily Romeis ◽  
Oriol Mitjà ◽  
Kelika A. Konda ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Vaccine Candidate ◽  
Treponema Pallidum ◽  
Phase Variation ◽  
Knowledge Gap ◽  
Transcription Level ◽  
Vaccine Candidates

AbstractBackgroundAn effective syphilis vaccine should elicit antibodies to Treponema pallidum subsp. pallidum (T. p. pallidum) surface antigens to induce pathogen clearance through opsonophagocytosis. Although the combination of bioinformatics, structural, and functional analyses of T. p. pallidum genes to identify putative outer membrane proteins (OMPs) resulted in a list of potential vaccine candidates, still very little is known about whether and how transcription of these genes is regulated during infection. This knowledge gap is a limitation to vaccine design, as immunity generated to an antigen that can be down-regulated or even silenced at the transcriptional level without affecting virulence would not induce clearance of the pathogen, hence allowing disease progression.Principal findingsWe report here that tp1031, the T. p. pallidum gene encoding the putative OMP and vaccine candidate TprL is differentially expressed in several T. p. pallidum strains, suggesting transcriptional regulation. Experimental identification of the tprL transcriptional start site revealed that a homopolymeric G sequence of varying length resides within the tprL promoter and that its length affects promoter activity compatible with phase variation. Conversely, in the closely related pathogen T. p. subsp. pertenue, the agent of yaws, where a naturally-occurring deletion has eliminated the tprL promoter region, elements necessary for protein synthesis, and part of the gene ORF, tprL transcription level are negligible compared to T. p. pallidum strains. Accordingly, the humoral response to TprL is absent in yaws-infected laboratory animals and patients compared to syphilis-infected subjects.ConclusionThe ability of T. p. pallidum to stochastically vary tprL expression should be considered in any vaccine development effort that includes this antigen. The role of phase variation in contributing to T. p. pallidum antigenic diversity should be further studied.Author SummarySyphilis is still an endemic disease in many low- and middle-income countries and has been resurgent in high-income nations for almost two decades now. In endemic areas, syphilis still causes significant morbidity and mortality in patients, particularly when its causative agent, the bacterium Treponema pallidum subsp. pallidum is transmitted to the fetus during pregnancy. Although there are significant ongoing efforts to identify an effective syphilis vaccine to bring into clinical trials within the decade in the U.S., such efforts are partially hindered by the lack of knowledge on transcriptional regulation of many genes encoding vaccine candidates. Here, we start addressing this knowledge gap for the putative outer membrane protein (OMP) and vaccine candidates TprL, encoded by the tp1031 gene. As we previously reported for other putative OMP-encoding genes of the syphilis agent, tprL transcription level appears to be affected by the length of a homopolymeric sequence of guanosines (Gs) located within the gene promoter. This is a mechanism known as phase variation and often involved in altering the surface antigenic profile of a bacterial pathogen to facilitate immune evasion and/or adaptation to the host milieu.

scholarly journals Production of Chlamydia pneumoniae Proteins in Bacillus subtilis and Their Use in Characterizing Immune Responses in the Experimental Infection Model

2003 ◽  
Vol 10 (3) ◽  
pp. 367-375 ◽  
Author(s):  
Ulla Airaksinen ◽  
Tuula Penttilä ◽  
Eva Wahlström ◽  
Jenni M. Vuola ◽  
Mirja Puolakkainen ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Large Scale ◽  
Chlamydia Pneumoniae ◽  
Signal Sequence ◽  
Expression Vectors ◽  
Infection Model

ABSTRACT Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious. To overcome these problems we produced chlamydial proteins in a heterologous host, Bacillus subtilis, a gram-positive nonpathogenic bacterium. The genes of Chlamydia pneumoniae major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the α-amylase gene from Bacillus amyloliquefaciens. C. pneumoniae genes were readily expressed in B. subtilis under the control of the α-amylase promoter. The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography. The Omp2 was separated as an insoluble fraction after 8 M urea treatment. The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test. The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of C. pneumoniae in mice. Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins.

scholarly journals Moraxella catarrhalis Outer Membrane Protein CD Elicits Antibodies That Inhibit CD Binding to Human Mucin and Enhance Pulmonary Clearance of M. catarrhalis in a Mouse Model

2007 ◽  
Vol 75 (6) ◽  
pp. 2818-2825 ◽  
Author(s):  
Dai-Fang Liu ◽  
John C. McMichael ◽  
Steven M. Baker
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Moraxella Catarrhalis ◽  
Signal Sequence ◽  
Vaccine Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Bacterial Clearance ◽  
Vitro Assay

ABSTRACT The outer membrane protein CD of Moraxella catarrhalis is considered to be a potential vaccine antigen against Moraxella infection. We purified the native CD from isolate O35E, administered it to mice, and detected considerable titers of anti-CD antibodies. Anti-CD sera were cross-reactive towards six different M. catarrhalis isolates and promoted bacterial clearance of O35E in a pulmonary challenge model. To circumvent the difficulty of generating large quantities of CD from M. catarrhalis for vaccine use, the CD gene from O35E was cloned into Escherichia coli, and the recombinant CD, expressed without a signal sequence or fusion tags, represented ∼70% of the total E. coli proteins. The recombinant CD formed inclusion bodies that were solubilized with 6 M urea and then purified by ion-exchange chromatography, a procedure that produced soluble CD of high purity and yield. Mice immunized with the purified recombinant CD had significant titers of anti-CD antibodies that were cross-reactive towards 24 different M. catarrhalis isolates. Upon challenge, these mice showed enhanced bacterial clearance of both O35E and a heterologous M. catarrhalis isolate, TTA24. In an in vitro assay, antisera to either the native or the recombinant CD inhibited the binding activity of CD to human tracheobronchial mucin in a serum concentration-dependent manner, and the extent of inhibition appeared to correlate with the corresponding anti-CD antibody titer and whole-cell enzyme-linked immunosorbent assay titer. Our results demonstrate that the recombinant CD is a promising vaccine candidate for preventing Moraxella infection.

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