Identification of B-cell linear epitopes in domains 1–3 of pyolysin of Trueperella pyogenes using polyclonal antibodies

Yang, Lei; Zhang, Yue; Wang, Haili; Ma, Bo; Xu, Liang; Wang, Junwei; Zhang, Wenlong

doi:10.1016/j.vetmic.2017.08.018

Cited by 8 publications

(9 citation statements)

References 24 publications

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“…In PLO, the homologous Asp and Asn are located at the 238th and 376th positions, respectively. The 238th Asp and 376th Asn are spatially close to our previously observed epitope [16]. …”

Section: Discussionsupporting

confidence: 78%

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Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin

Zhang

Wang

et al. 2018

Virulence

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Trueperella pyogenes (T. pyogenes) is an important opportunistic pathogen. Pyolysin (PLO) importantly contributes to the pathogenicity of T. pyogenes. However, the relationship between the structure and function and the virulence of PLO is not well documented. In the current study, recombinant PLO (rPLO) and three rPLO mutants were prepared. rPLO D238R, a mutant with the 238th aspartic acid replaced with an arginine, showed impairment in oligomerization activity on cholesterol-containing liposome and pore-forming activity on sheep red blood cell membrane. Further study employing the prepared mutants confirmed that the pore-forming activity of PLO is essential for inducing excessive inflammation responses in mice by upregulating the expression levels of IL-1β, TNF-α, and IL-6. By contrast, rPLO P499F, another mutant with impaired cell membrane binding capacity, elicited an inflammation response that was dependent on pathogen-associated molecular pattern (PAMP) activity, given that the mutant significantly upregulated the expression of IL-10 in macrophages and in mice, whereas rPLO did not. Results indicated that domain 1 of the PLO molecule plays an important role in maintaining pore-forming activity. Moreover, the PLO pore-forming activity and not PAMP activity is responsible for the inflammation-inducing effect of PLO. The results of this study provided new information for research field on the structure, function, and virulence of PLO.Abbreviations: T. pyogenes: Trueperella pyogenes; PLO: Pyolysin; rPLO: recombinant PLO; PAMP: pathogen-associated molecular pattern; CDCs: cholesterol-dependent cytolysins; PLY: pneumolysin; NLRP3: NLR family pyrin domain containing protein 3; PRRs: pattern recognition receptors; Asp: aspartic acid; TLR4: Toll-like receptor 4; Arg: arginine; Asn: asparagine; IPTG: Isopropyl-β-d-thiogalactoside; PBS: phosphate-buffered saline; sRBCs: sheep red blood cells; TEM: Transmission electron microscopy; RBCM: red blood cell membrane; SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; NC membrane: nitrocellulose membrane; SDS-AGE: dodecyl sulfate agarose gel electrophoresis; MDBK cells: Madin–Darby bovine kidney cells; RPMI-1640 medium: Roswell Park Memorial Institute-1640 medium; FBS: fetal bovine serum; BMDMs: bone marrow-derived macrophages; TNF-α: tumor necrosis factor α; IL-1β: interleukin-1β; IFN-γ: interferon-γ; TGF-β: transforming growth factor-β; ELISA: enzyme-linked immunosorbent assay

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“…In PLO, the homologous Asp and Asn are located at the 238th and 376th positions, respectively. The 238th Asp and 376th Asn are spatially close to our previously observed epitope [16]. …”

Section: Discussionsupporting

confidence: 78%

“…However, D1 in CDCs is not extensively characterized. One of our previous studies showed that antibodies targeted to epitopes located in D1 of PLO exhibit antihemolytic activities [16]. On the basis of this finding, we speculated that D1 played an essential role in forming pores, at least for PLO.…”

Section: Discussionmentioning

confidence: 78%

Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin

Zhang

Wang

et al. 2018

Virulence

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“…Another piece of evidence that supports the above speculation is that the level of PLO-specific IgG of mice that received sRBCM-trapped tPLOA2 was substantially lower than that of mice that received sRBCM-trapped tPLOA1 (Figure 4). Amino acid segments 1-110 and 190-296 are located at the D2 and D3 of the PLO molecule, respectively [27]. The D3 of CDC molecules forms the inner side of membrane-penetrating pores after an entirely structural refolding, and the D2 of CDC molecules undergoes a simple rotation and forms the outer side of the pores when the prepore complexes transform to pores [28].…”

Section: Discussionmentioning

confidence: 99%

“…The 1-110 and 190-296 segments of the PLO molecule contain several linear epitopes [27]; hence, we speculated that the recombinant proteins involving the two segments and D4 of PLO may be promising antigens for T. pyogenes vaccines. The recombinant antigen molecules may not have the biological functions of the intact PLO molecules and thereby show good safety.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the Potency of Two Pyolysin-Derived Recombinant Proteins as Vaccine Candidates of Trueperella Pyogenes in a Mouse Model: Pyolysin Oligomerization and Structural Change Affect the Efficacy of Pyolysin-Based Vaccines

et al. 2020

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Trueperella pyogenes (T. pyogenes) is an important opportunistic pathogen in livestock and wild animals. However, only one commercial T. pyogenes vaccine is currently available, and its immunoprotective effect is not ideal. Pyolysin (PLO) is one of the important virulence factors expressed by T. pyogenes and one of the targets for the development of new T. pyogenes vaccines. In this study, we constructed two recombinant antigens, tPLOA1 (contains amino acids 1–110 and domain 4 of the PLO molecule) and tPLOA2 (contains amino acids 190–296 and domain 4 of the PLO molecule). Vaccines were prepared by mixing the two recombinant antigens with incomplete Freund’s adjuvant or sheep red blood cell membrane and provided partial immune protection to immunized mice against the lethal challenge of T. pyogenes. Analysis of the PLO-specific IgG levels of immunized mice indicated that the antibody-inducing potency and immunoprotective efficacy of PLO-based vaccines are affected by the oligomerization and structural changes of PLO after binding to a cholesterol-containing membrane. In addition, the titer of anti-hemolysis antibodies is not a suitable indicator of the immunoprotective effect of these vaccines in PLO-based vaccine-immunized animals. The results provide new insights into the development of T. pyogenes vaccines.

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“…Trueperella pyogenes (formerly Arcanobacterium pyogenes ) is a Gram-positive, nonmotile, nonspore-forming, irregular rod. This bacterium is a common inhabitant of mucous membranes of the upper respiratory, gastrointestinal, and urogenital tracts of animals [ 1 , 2 , 3 ]. However, T. pyogenes can be pathogenic for both domestic and wild animals and causes different diseases, including mastitis, metritis, balanoposthitis, pneumonia, and abscesses in various organs and tissues [ 1 , 3 , 4 , 5 ].…”

Section: Introductionmentioning

confidence: 99%

New Determinants of Aminoglycoside Resistance and Their Association with the Class 1 Integron Gene Cassettes in Trueperella pyogenes

Kwiecień

Stefańska

Chrobak-Chmiel

et al. 2020

IJMS

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Trueperella pyogenes is an important opportunistic animal pathogen. Different antimicrobials, including aminoglycosides, are used to treat T. pyogenes infections. The aim of the present study was to evaluate aminoglycoside susceptibility and to detect aminoglycoside resistance determinants in 86 T. pyogenes isolates of different origin. Minimum inhibitory concentration of gentamicin, streptomycin, and kanamycin was determined using a standard broth microdilution method. Genetic elements associated with aminoglycoside resistance were investigated by PCR and DNA sequencing. All studied isolates were susceptible to gentamicin, but 32.6% and 11.6% of them were classified as resistant to streptomycin and kanamycin, respectively. A total of 30 (34.9%) isolates contained class 1 integrons. Class 1 integron gene cassettes carrying aminoglycoside resistance genes, aadA11 and aadA9, were found in seven and two isolates, respectively. Additionally, the aadA9 gene found in six isolates was not associated with mobile genetic elements. Moreover, other, not carried by gene cassettes, aminoglycoside resistance genes, strA-strB and aph(3’)-IIIa, were also detected. Most importantly, this is the first description of all reported genes in T. pyogenes. Nevertheless, the relevance of the resistance phenotype to genotype was not perfectly matched in 14 isolates. Therefore, further investigations are needed to fully explain aminoglycoside resistance mechanisms in T. pyogenes.

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Identification of B-cell linear epitopes in domains 1–3 of pyolysin of Trueperella pyogenes using polyclonal antibodies

Cited by 8 publications

References 24 publications

Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin

Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin

Evaluation of the Potency of Two Pyolysin-Derived Recombinant Proteins as Vaccine Candidates of Trueperella Pyogenes in a Mouse Model: Pyolysin Oligomerization and Structural Change Affect the Efficacy of Pyolysin-Based Vaccines

New Determinants of Aminoglycoside Resistance and Their Association with the Class 1 Integron Gene Cassettes in Trueperella pyogenes

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