The pyruvate dehydrogenase complex of Mycoplasma hyopneumoniae contains a novel lipoyl domain arrangement

Matic, Jake N.; Wilton, Jody L.; Towers, Rebecca J.; Scarman, Anthony L.; Minion, F. Chris; Walker, Mark J.; Djordjevic, Steven P.

doi:10.1016/s0378-1119(03)00798-4

Cited by 19 publications

(13 citation statements)

References 29 publications

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“…4). Structural-homology searches of Lpd in other sequenced mycoplasma and nonmycoplasma species, such as Mycoplasma pneumoniae, Mycoplasma hyopneumoniae, Mycoplasma genitalium, Mycoplasma penetrans, Mycoplasma pulmonis, Pseudomonas fluorescens, Azotobacter vinelandii, and Neisseria meningitidis (4,7,13,20,27,28,29,38), revealed that this disulfide oxidoreductase has a redoxactive site and a NAD/FAD binding domain within the intracellular amino-terminal portion of the polypeptide sequence. It also has a highly conserved dimerization domain located in its intracellular carboxyl-terminal portion.…”

Section: Resultsmentioning

confidence: 99%

“…Sterile 250-ml Erlenmeyer flasks containing 69 ml of Hayflick's broth were inoculated simultaneously with 1 ml of equal numbers of pretitered (based on previous optical-density values at 620 nm [OD 620 ]) M. gallisepticum R low and each of the individual mutant stock cultures. The cells were incubated at 37°C in a 250-rpm orbital shaking incubator and were harvested at 2, 4,6,8,10,12,14,16,28,32,40,49, and 54 hours postinoculation. The growth was measured spectrophotometrically by assessing Animals.…”

Section: Methodsmentioning

confidence: 99%

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Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase ( lpd ), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants

et al. 2006

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To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.Mycoplasma gallisepticum is the primary etiologic agent of the chronic respiratory disease complex in chickens and infectious sinusitis in turkeys. Primary inflammatory responses in the respiratory tract associated with infection are sinusitis, tracheitis, bronchitis, and airsacculitis. Colonization of the respiratory tract leads to ciliostasis and deciliation of the tracheal epithelium, allowing secondary infection by other bacterial and viral pathogens, such as Newcastle disease virus, infectious bronchitis virus, and Escherichia coli (24). Common signs of M. gallisepticum infection include nasal discharge, tracheal rales, weight loss, and decreased egg production. Mycoplasma gallisepticum is highly contagious in commercial chicken and turkey flocks, spreading horizontally in populations through aerosol, dust, and feathers and vertically transmitted through eggs (8,10,24,40). Its economic impact on the poultry industry is significant, leading to millions of dollars of loss each year. Efforts to improve disease prevention and control programs require increased research to identify novel virulenceassociated determinants. These determinants may provide the basis for new and promising vaccines or targets for other antimicrobial ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase ( lpd ), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants

et al. 2006

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“…The PDH complex converts pyruvate to acetyl coenzyme A, which is subsequently used in the tricarboxylic acid cycle to generate NADH, ATP, and reduced flavin adenine dinucleotide (7). The PDH complex is known to be highly immunogenic in other bacterial species, such as Neisseria meningitidis (1), Mycoplasma capricolum (40), and Mycoplasma hyopneumoniae (28). Recently, PDH has been tested as a DNA vaccine against Mycoplasma mycoides subsp.…”

Section: Discussionmentioning

confidence: 99%

Proteomic Profiling and Identification of Immunodominant Spore Antigens of Bacillus anthracis , Bacillus cereus , and Bacillus thuringiensis

DelVecchio

Connolly

Alefantis

et al. 2006

Appl Environ Microbiol

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Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, ⌬-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid-and immuno-based detection systems as well as next-generation vaccine development.

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“…Uniquely, this clone lacks pdhD upstream DNA (likely to contain a promoter region), which would agree with the hypothesis that P57 is derived from the pdhD gene product. The Pdh complex, particularly the lipoyl-binding domain, is known to be highly immunogenic in other bacterial species (e.g., Neisseria meningitidis [1], Mycoplasma capricolum [43], and Mycoplasma hyopneumoniae [24]). In M. mycoides subsp.…”

Section: Discussionmentioning

confidence: 99%

Phage Library Screening for the Rapid Identification and In Vivo Testing of Candidate Genes for a DNA Vaccine against Mycoplasma mycoides subsp. mycoides Small Colony Biotype

et al. 2006

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A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage ZAP Express vector which contains both prokaryotic (P lac ) and eukaryotic (P CMV ) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which P CMV -driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into ZAP Express, and two strongly immunodominant clones, -A8 and -B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone -A8 expressed an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone -B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones -A8 and -B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone -A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone -A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.Whole bacteriophage (phage) particles have recently been described as highly efficient DNA vaccine delivery vehicles (6,7,17,20), inducing significant antibody responses in both laboratory mice (7) and larger animals such as rabbits (20) and sheep (33). Bacteriophages are viruses of bacteria and are metabolically inert, requiring the host bacterium for growth and propagation. Using standard "naked" DNA vaccination, purified genetic material in the form of a plasmid encoding protective antigens is given to the host and used to stimulate an immune response and give protection against pathogens (37,42). With bacteriophage genetic immunization, expression cassettes containing a vaccine gene under the control of a su...

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The pyruvate dehydrogenase complex of Mycoplasma hyopneumoniae contains a novel lipoyl domain arrangement

Cited by 19 publications

References 29 publications

Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase ( lpd ), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants

Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase ( lpd ), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants

Proteomic Profiling and Identification of Immunodominant Spore Antigens of Bacillus anthracis , Bacillus cereus , and Bacillus thuringiensis

Phage Library Screening for the Rapid Identification and In Vivo Testing of Candidate Genes for a DNA Vaccine against Mycoplasma mycoides subsp. mycoides Small Colony Biotype

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