Immunogenicity and reactivity of novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and conserved MAP1156 proteins with sera from experimentally and naturally infected animals

John P. Bannantine, Avery L. Paulson, Ofelia Chacon, Robert J. Fenton, Denise K. Zinniel, David S. McVey, David R. Smith, Charles J. Czuprynski, Raul G Barletta

Research output: Contribution to journalArticle

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Abstract

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5′ and 3′ regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein (P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis-infected cattle mount a humoral response to both MAP1152 and MAP1156 crossreactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD.

Original languageEnglish (US)
Pages (from-to)105-112
Number of pages8
JournalClinical and Vaccine Immunology
Volume18
Issue number1
DOIs
StatePublished - Jan 1 2011

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Paratuberculosis
Mycobacterium avium
Blood Proteins
Animals
Maltose-Binding Proteins
Proteins
Antigens
Serum
Assays
Fusion reactions
Genes
Diacylglycerol O-Acyltransferase
Recombinant Proteins
Recombinant proteins
Immunosorbents
Rabbits
Metabolism
Escherichia coli
Epitopes
Triglycerides

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Clinical Biochemistry
  • Microbiology (medical)

Cite this

Immunogenicity and reactivity of novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and conserved MAP1156 proteins with sera from experimentally and naturally infected animals. / Bannantine, John P.; Paulson, Avery L.; Chacon, Ofelia; Fenton, Robert J.; Zinniel, Denise K.; McVey, David S.; Smith, David R.; Czuprynski, Charles J.; Barletta, Raul G.

In: Clinical and Vaccine Immunology, Vol. 18, No. 1, 01.01.2011, p. 105-112.

Research output: Contribution to journalArticle

Bannantine, John P. ; Paulson, Avery L. ; Chacon, Ofelia ; Fenton, Robert J. ; Zinniel, Denise K. ; McVey, David S. ; Smith, David R. ; Czuprynski, Charles J. ; Barletta, Raul G. / Immunogenicity and reactivity of novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and conserved MAP1156 proteins with sera from experimentally and naturally infected animals. In: Clinical and Vaccine Immunology. 2011 ; Vol. 18, No. 1. pp. 105-112.
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abstract = "Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5′ and 3′ regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein (P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis-infected cattle mount a humoral response to both MAP1152 and MAP1156 crossreactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD.",
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