scholarly journals Construction of Mycoplasma hyopneumoniae P97 Null Mutants

2021 ◽  
Vol 12 ◽  
Author(s):  
Jeannett M. Clampitt ◽  
Melissa L. Madsen ◽  
F. Chris Minion
Keyword(s):  
Monoclonal Antibodies ◽  
Reca Protein ◽  
Gene Families ◽  
Mycoplasma Hyopneumoniae ◽  
Surface Proteins ◽  
Production Environment ◽  
Cell Adherence ◽  
Pk15 Cell ◽  
Characteristic Banding ◽  
Null Mutants

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia, a world-wide problem in the pig industry. This disease is characterized by a dry, non-productive cough, labored breathing, and pneumonia. Despite years of research, vaccines are marginally effective, and none fully protect pigs in a production environment. A better understanding of the host-pathogen interactions of the M. hyopneumoniae-pig disease, which are complex and involve both host and pathogen components, is required. Among the surface proteins involved in virulence are members of two gene families called P97 and P102. These proteins are the adhesins directing attachment of the organism to the swine respiratory epithelium. P97 is the major ciliary binding adhesin and has been studied extensively. Monoclonal antibodies that block its binding to swine cilia have contributed extensively to its characterization. In this study we use recombination to construct null mutants of P97 in M. hyopneumoniae and characterize the resulting mutants in terms of loss of protein by immunoblot using monoclonal antibodies, ability to bind purified swine cilia, and adherence to PK15 cells. Various approaches to recombination with this fastidious mycoplasma were tested including intact plasmid DNA, single-stranded DNA, and linear DNA with and without a heterologous RecA protein. Our results indicate that recombination can be used to generate site-specific mutants in M. hyopneumoniae. P97 mutants are deficient in cilia binding and PK15 cell adherence, and lack the characteristic banding pattern seen in immunoblots developed with the anti-P97 monoclonal antibody.

scholarly journals Survey of Surface Proteins from the Pathogenic Mycoplasma hyopneumoniae Strain 7448 Using a Biotin Cell Surface Labeling Approach

PLoS ONE ◽  
2014 ◽  
Vol 9 (11) ◽  
pp. e112596 ◽  
Author(s):  
Luciano Antonio Reolon ◽  
Carolina Lumertz Martello ◽  
Irene Silveira Schrank ◽  
Henrique Bunselmeyer Ferreira
Keyword(s):  
Cell Surface ◽  
Mycoplasma Hyopneumoniae ◽  
Surface Proteins ◽  
Labeling Approach

Monoclonal antibodies against surface antigens of schistosomula of Schistosoma mansoni

Parasitology ◽  
1982 ◽  
Vol 84 (1) ◽  
pp. 65-82 ◽  
Author(s):  
D. W. Taylor ◽  
A. F. Butterworth
Keyword(s):  
Monoclonal Antibodies ◽  
Gel Electrophoresis ◽  
Primary Infection ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Surface Antigens ◽  
Soft Agar ◽  
Surface Proteins ◽  
Spleen Cells

SUMMARYMonoclonal antibodies have been produced after fusion of NS-1 murine myeloma cells with spleen cells from mice immunized either by chronic primary infection or with irradiated cercariae: in both cases, animals were challenged with live cercariae 7 days before fusion. The initial cultures were screened for anti-schistosomular antibodies both by a radioimmunoassay with whole schistosomulum extracts and by immunofluorescence. There was no correlation between the two techniques and subsequent screening was carried out by immunofluorescence. Cloning was carried out in soft agar and 7 cloned cell lines, from 5 initial cultures, were selected for detailed study. Products of 6 of these 7 lines were monoclonal, as judged by isoelectricfocusing of [35S]methionine-labelled supernatant fluids, and their binding to live schistosomula was specific. None of the antibodies showed detectable activity in mediating eosinophil- or complement-dependent damage to schistosomula in vitro. However, 2 antibodies were successfully used to isolate surface proteins with an apparent molecular weight of 24000 on SDS-polyacrylamide gel electrophoresis.

Sequences of the variable regions of three monoclonal antibodies specific for histidine-containing protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system

1991 ◽  
Vol 69 (4) ◽  
pp. 297-302 ◽  
Author(s):  
Teresa Steeves ◽  
M. Michele Barry ◽  
Harry W. Duckworth ◽  
E. Bruce Waygood ◽  
Jeremy S. Lee
Keyword(s):  
Monoclonal Antibodies ◽  
Gene Sequencing ◽  
Gene Families ◽  
Gene Sequences ◽  
Phosphotransferase System ◽  
Variable Regions ◽  
Gene Usage ◽  
Vh Gene

The variable regions of three monoclonal antibodies, Jel 42, Jel 44, and Jel 324, specific for the histidine-containing protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system have been sequenced from their respective mRNAs. The Vh gene families were deduced from the percent homology to the concensus gene sequences and the J gene and D gene usage was also analysed.Key words: monoclonal antibodies, gene sequencing.

scholarly journals The Genome Sequence of Mycoplasma hyopneumoniae Strain 232, the Agent of Swine Mycoplasmosis

2004 ◽  
Vol 186 (21) ◽  
pp. 7123-7133 ◽  
Author(s):  
F. Chris Minion ◽  
Elliot J. Lefkowitz ◽  
Melissa L. Madsen ◽  
Barbara J. Cleary ◽  
Steven M. Swartzell ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Antigenic Variation ◽  
Gene Families ◽  
Mycoplasma Hyopneumoniae ◽  
23S Rrna ◽  
Hypothetical Proteins ◽  
Protein Coding ◽  
Coding Sequences ◽  
Phase Switching ◽  
Transporter Family

ABSTRACT We present the complete genome sequence of Mycoplasma hyopneumoniae, an important member of the porcine respiratory disease complex. The genome is composed of 892,758 bp and has an average G+C content of 28.6 mol%. There are 692 predicted protein coding sequences, the average protein size is 388 amino acids, and the mean coding density is 91%. Functions have been assigned to 304 (44%) of the predicted protein coding sequences, while 261 (38%) of the proteins are conserved hypothetical proteins and 127 (18%) are unique hypothetical proteins. There is a single 16S-23S rRNA operon, and there are 30 tRNA coding sequences. The cilium adhesin gene has six paralogs in the genome, only one of which contains the cilium binding site. The companion gene, P102, also has six paralogs. Gene families constitute 26.3% of the total coding sequences, and the largest family is the 34-member ABC transporter family. Protein secretion occurs through a truncated pathway consisting of SecA, SecY, SecD, PrsA, DnaK, Tig, and LepA. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The DnaK-DnaJ-GrpR complex is intact, providing the only control over protein folding. There are several proteases that might serve as virulence factors, and there are 53 coding sequences with prokaryotic lipoprotein lipid attachment sites. Unlike other mycoplasmas, M. hyopneumoniae contains few genes with tandem repeat sequences that could be involved in phase switching or antigenic variation. Thus, it is not clear how M. hyopneumoniae evades the immune response and establishes a chronic infection.

scholarly journals Inhibition ofBordetella pertussisfilamentous hemagglutinin-mediated cell adherence with monoclonal antibodies

1993 ◽  
Vol 106 (1) ◽  
pp. 31-38 ◽  
Author(s):  
Elizabeth Leininger ◽  
Peter G. Probst ◽  
Michael J. Brennan ◽  
James G. Kenimer
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Adherence

scholarly journals Characterization of a Multigene Family Undergoing High-Frequency DNA Rearrangements and Coding for Abundant Variable Surface Proteins in Mycoplasma agalactiae

2000 ◽  
Vol 68 (8) ◽  
pp. 4539-4548 ◽  
Author(s):  
M. D. Glew ◽  
L. Papazisi ◽  
F. Poumarat ◽  
D. Bergonier ◽  
R. Rosengarten ◽  
...  
Keyword(s):  
Phase Variation ◽  
Gene Families ◽  
Surface Proteins ◽  
Dna Rearrangements ◽  
Reading Frame ◽  
Mycoplasma Agalactiae ◽  
Amino Terminal ◽  
Clonal Variant ◽  
Variable Surface

ABSTRACT A family of abundant surface proteins (Vpmas [variable proteins ofMycoplasma agalactiae]) undergoing phase variation inM. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboringvpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes,vpmaX and vpmaY, were orientated divergently and shared highly homologous 5′ untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. ThevpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. ThevpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by thevsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.

scholarly journals Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools

2016 ◽  
Vol 82 (17) ◽  
pp. 5465-5476 ◽  
Author(s):  
Cathy X. Y. Zhang ◽  
Brian W. Brooks ◽  
Hongsheng Huang ◽  
Franco Pagotto ◽  
Min Lin
Keyword(s):  
Monoclonal Antibodies ◽  
Listeria Monocytogenes ◽  
Surface Protein ◽  
Protein Detection ◽  
Food Samples ◽  
Surface Proteins ◽  
Content Type ◽  
Selective Enrichment ◽  
Wide Range ◽  
Serotype 4B

ABSTRACTThe Gram-positive bacteriumListeria monocytogenescauses a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range ofL. monocytogenesserotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of theL. monocytogenesserotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains ofL. monocytogeneswhich are variable among otherListeriaspecies. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46L. monocytogeneslineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17L. monocytogeneslineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some otherListeriaspecies grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker ofL. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples.IMPORTANCEL. monocytogenesis traditionally divided into at least 12 serotypes. Currently, there are no monoclonal antibodies (MAbs) available that are capable of binding to the surface ofL. monocytogenesstrains representing all 12 serotypes. Such antibodies would be useful and are needed for the development of methods to detect and isolateL. monocytogenesfrom food samples. In our study, we aimed to identify surface proteins that possess regions of well-conserved amino acid sequences among various serotypes and then to employ them as antigen targets (biomarkers) for the development of MAbs. Through bioinformatics and protein expression analysis, we identified one of the four putative surface protein candidates, LMOf2365_0639, encoded by the genome of theL. monocytogenesserotype 4b strain F2365, as a useful surface biomarker. Extensive assessment of 35 MAbs raised against LMOf2365_0639 in our study revealed three MAbs (M3643, M3644, and M3651) that recognized a wide range ofL. monocytogenesisolates.

scholarly journals Functional Properties of Borrelia burgdorferi recA

2004 ◽  
Vol 186 (8) ◽  
pp. 2275-2280 ◽  
Author(s):  
Dionysios Liveris ◽  
Vishwaroop Mulay ◽  
Ira Schwartz
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Uv Irradiation ◽  
Reca Protein ◽  
Primary Role ◽  
Rapid Loss ◽  
E Coli ◽  
Uv Dose ◽  
Null Mutants

ABSTRACT Functions of the Borrelia burgdorferi RecA protein were investigated in Escherichia coli recA null mutants. Complementation with B. burgdorferi recA increased survival of E. coli recA mutants by 3 orders of magnitude at a UV dose of 2,000 μJ/cm2. The viability at this UV dose was about 10% that provided by the homologous recA gene. Expression of B. burgdorferi recA resulted in survival of E. coli at levels of mitomycin C that were lethal to noncomplemented hosts. B. burgdorferi RecA was as effective as E. coli RecA in mediating homologous recombination in E. coli. Furthermore, E. coli λ phage lysogens complemented with B. burgdorferi recA produced phage even in the absence of UV irradiation. The level of phage induction was 55-fold higher than the level in cells complemented with the homologous recA gene, suggesting that B. burgdorferi RecA may possess an enhanced coprotease activity. This study indicates that B. burgdorferi RecA mediates the same functions in E. coli as the homologous E. coli protein mediates. However, the rapid loss of viability and the absence of induction in recA expression after UV irradiation in B. burgdorferi suggest that recA is not involved in the repair of UV-induced damage in B. burgdorferi. The primary role of RecA in B. burgdorferi is likely to be a role in some aspect of recombination.

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