Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display

Ribeiro, Eduardo Lara; Fujimura, Patrícia Tiemi; Ueira-Vieira, Carlos; Goulart, Luiz Ricardo; Alves, Telma Maria; Lage, Andrey Pereira; Heinemann, Marcos Bryan

doi:10.1590/0103-8478cr20200945

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Microbiology • Cienc. Rural 51 (8) • 2021 • https://doi.org/10.1590/0103-8478cr20200945 copy

Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display

Seleção e caracterização de peptídeos miméticos a Campylobacter fetus subsp. venerealis usando phage display

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT:

Bovine genital campylobacteriosis (BGC) is a venereal and subclinical disease that affects the fertility of cattle herds, and it is caused by Campylobacter fetus subsp. venerealis . This study selected peptides mimetic to the BGC-causing agent from a phage library. Phage display is a technique that applies bacteriophage libraries that reveal peptides fused to the viral capsid in biological selections against target proteins. Biopannings were performed for biological selection in the phage library using rabbit hyperimmune serum and C. fetus subsp. venerealis protein extract. Five selected heptapeptides were considered mimetic to Cfv-NCTC 10354 based on the results of bioinformatics analysis and assays with hyperimmune serum and cervicovaginal mucus obtained from heifers. ALASLPL and LSYLFPP were the most reactive peptides and considered promising as possible mimetic immunogens for C. fetus subsp. venerealis.

Key words:
Campylobacteriosis; phage display; peptides

Authorship

Eduardo Lara Ribeiro

Prefeitura Municipal de Belo Horizonte, Distrito Regional de Venda Nova, Gerência de Zoonoses, Belo Horizonte, MG, Brasil.Prefeitura Municipal de Belo HorizonteBrazilBelo Horizonte, MG, BrazilPrefeitura Municipal de Belo Horizonte, Distrito Regional de Venda Nova, Gerência de Zoonoses, Belo Horizonte, MG, Brasil.

http://orcid.org/0000-0001-8012-8810

Patrícia Tiemi Fujimura

Instituto de Genética e Bioquímica, Laboratório de Nanobiotecnologia, Universidade Federal de Uberlândia (UFU), Uberlândia, MG, Brasil.Universidade Federal de Uberlândia (UFU),BrazilUberlândia, MG, BrazilInstituto de Genética e Bioquímica, Laboratório de Nanobiotecnologia, Universidade Federal de Uberlândia (UFU), Uberlândia, MG, Brasil.

http://orcid.org/0000-0002-9048-1877

Carlos Ueira-Vieira

http://orcid.org/0000-0002-8369-9069

Luiz Ricardo Goulart

http://orcid.org/0000-0002-1803-4861

Telma Maria Alves

Ceva Veterinária, Departamento de GIPID, Juatuba, MG, Brasil.Ceva VeterináriaBrasilJuatuba, MG, BrasilCeva Veterinária, Departamento de GIPID, Juatuba, MG, Brasil.

http://orcid.org/0000-0002-6412-9204

Andrey Pereira Lage

Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais (UFMG),BrazilBelo Horizonte, MG, BrazilDepartamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brasil.

http://orcid.org/0000-0002-9325-5728

Marcos Bryan Heinemann ^*E-mail: marcosbryan@usp.br. *Corresponding author

Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo (USP), 05508-270, São Paulo, SP, Brasil. Universidade de São Paulo (USP)BrazilSão Paulo, SP, BrazilDepartamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo (USP), 05508-270, São Paulo, SP, Brasil.

http://orcid.org/0000-0001-6444-9788

E-mail: marcosbryan@usp.br. ^*Corresponding author

DECLARTION OF CONFLICT OF INTEREST

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

SCIMAGO INSTITUTIONS RANKINGS

Ceva Veterinária, Departamento de GIPID, Juatuba, MG, Brasil.Ceva VeterináriaBrasilJuatuba, MG, BrasilCeva Veterinária, Departamento de GIPID, Juatuba, MG, Brasil.

Figures | Tables

Figures (6)
Tables (1)

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Figure 1
Summary flowchart of complete biopanning selection cycle in six steps. 1) coupling of rabbit IgG in magnetic microspheres; 2) interaction between phage library and magnetic microspheres; 3) negative dissociation with rabbit pre-immune serum; 4) positive dissociation with bacterial proteins; 5) incubation of the selected phage in E. coli inoculum; 6) precipitation of the amplified phages.

Thumbnail

Figure 2
Phage-ELISA involving 12 selected phages and wild phage M13 with hyperimmune rabbit serum against C. fetus subsp. venerealis and preimmune serum. Comparison of the absorbance (OD 490nm) means of the reactions of each phage indicate statistically significant difference (^*) based on ANOVA and the Student’s t-test (p < 0.05).

Thumbnail

Figure 3
Indirect ELISA involving five synthetic peptides, bacterial extract of C. fetus subsp. venerealis (Cfv) and negative control (BSA) with preimmune and hyperimmune rabbit serum. Comparison of the absorbance (OD 490nm) means of reactions of each peptide indicates statistically significant difference (*) based on ANOVA and Student’s t-test (p < 0.05).

Thumbnail

Figure 4
Indirect ELISA involving synthetic peptides and cervicovaginal mucus positive and negative to BGC. The positive control is represented by C. fetus subsp. venerealis bacterial extract (Cfv) and negative control by BSA. Comparison of the absorbance (OD490 nm) means of the reactions of each peptide and controls with each type of mucus sample and within peptides indicates a statistically significant difference (^*) based on ANOVA and the Student’s t-test (p < 0.05).

Thumbnail

Figure 5
Ratio between positive (EI +) and negative (EI-) ELISA indexes obtained from indirect ELISA. Synthetic peptides were tested with rabbit hyperimmune serum and cervicovaginal mucus. Control is represented by C. fetus subsp. venerealis bacterial extract (Cfv) and negative control by BSA.

Thumbnail

Figure 6
Most relevant amino acid similarities between the selected peptides and Cfv-NCTC 10354 proteins. Accession numbers on GenBank (Taxid: 983328).

Thumbnail

Table 1
Selected heptapeptides from phage library with Cfv-NCTC 10354.

Figure 1 Summary flowchart of complete biopanning selection cycle in six steps. 1) coupling of rabbit IgG in magnetic microspheres; 2) interaction between phage library and magnetic microspheres; 3) negative dissociation with rabbit pre-immune serum; 4) positive dissociation with bacterial proteins; 5) incubation of the selected phage in E. coli inoculum; 6) precipitation of the amplified phages.

Figure 2 Phage-ELISA involving 12 selected phages and wild phage M13 with hyperimmune rabbit serum against C. fetus subsp. venerealis and preimmune serum. Comparison of the absorbance (OD 490nm) means of the reactions of each phage indicate statistically significant difference (^*) based on ANOVA and the Student’s t-test (p < 0.05).

Figure 3 Indirect ELISA involving five synthetic peptides, bacterial extract of C. fetus subsp. venerealis (Cfv) and negative control (BSA) with preimmune and hyperimmune rabbit serum. Comparison of the absorbance (OD 490nm) means of reactions of each peptide indicates statistically significant difference (*) based on ANOVA and Student’s t-test (p < 0.05).

Figure 4 Indirect ELISA involving synthetic peptides and cervicovaginal mucus positive and negative to BGC. The positive control is represented by C. fetus subsp. venerealis bacterial extract (Cfv) and negative control by BSA. Comparison of the absorbance (OD490 nm) means of the reactions of each peptide and controls with each type of mucus sample and within peptides indicates a statistically significant difference (^*) based on ANOVA and the Student’s t-test (p < 0.05).

Figure 5 Ratio between positive (EI +) and negative (EI-) ELISA indexes obtained from indirect ELISA. Synthetic peptides were tested with rabbit hyperimmune serum and cervicovaginal mucus. Control is represented by C. fetus subsp. venerealis bacterial extract (Cfv) and negative control by BSA.

Figure 6 Most relevant amino acid similarities between the selected peptides and Cfv-NCTC 10354 proteins. Accession numbers on GenBank (Taxid: 983328).

Table 1 Selected heptapeptides from phage library with Cfv-NCTC 10354.

Selected heptapeptides from phage library^*	Frequency^**
ALASLPL	2.27%
HVPGALF	2.27%
LFSPLSA	6.81%
LSYLFPP	22.72%
PAPFHVR	4.54%
PFASFLS	34.09%
QTLSRPS	1.13%
SLSPVNS	17.04%
SSAHSYF	3.40%
TLPYPTQ	2.27%
TPLLSPF	2.27%
VTTAPFT	1.13%

^*Peptides were named with seven letters, each an abbreviation of an amino acid: A - Alanine; F - Phenylalanine; G - Glycine; H - Histidine; L - Leucine; N - Asparagine; P - Proline; Q - Glutamine; R - Arginine; S - Serine; T - Threonine; V -Valine; Y - Tyrosine (LEHNINGER et al., 2014LEHNINGER, A. L. et al. Princípios de Bioquímica. 6 ed. Artmed, Porto Alegre, 2014.):
^**Frequencies were calculated from 88 selected phage clones.

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Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display

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[1] E-mail: marcosbryan@usp.br. ^*Corresponding author