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O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Nandakumar S, Kannanganat S, Dobos KM, Lucas M, Spencer JS, Fang S, McDonald MA, Pohl J, Birkness K, Chamcha V, Ramirez MV, Plikaytis BB, Posey JE, Amara RR, Sable SB - PLoS Pathog. (2013)

Bottom Line: Glycosylation is the most abundant post-translational polypeptide chain modification in nature.Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood.These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Tuberculosis Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.

ABSTRACT
Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

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T cell reactivity to in vitro-mannosylated rApa C-terminal.(A) Pooled splenocytes from BCG infected mice (n = 4) were stimulated in vitro with nApa, rApa or 32 individual non-glycosylated synthetic Apa peptides in ELISPOT assay. IFN-γ SFU/106 cells at 12 and 32 wk time points are shown. (B–C) Synthetic C-terminal glycopeptide (Gp281-325)- or control non-glycopeptide (NGp281-325)-specific T cell response (indicated inside dotted box) in mice. Stimulation with proteins (nApa or rApa) or non-glycopeptides (p271-288 or p301-320) was also included as controls. (B) Ag- or peptide-specific IFN-γ, IL-17 or IL-4 SFU/106 pooled lung or spleen cells of BCG or Mtb infected mice at 32 and 26 wks, respectively. (C) The proportions of peptide-specific 1+, 2+ and 3+ cytokine producing T cell subsets constituting total cytokine positive (+) CD4+ T cells in the spleen of BCG infected mice at 32 wks, expressed as % of CD4+ T cells. Data in A–C are means ± s.e.m. *Significant using 1-way ANOVA followed by Bonferroni's test.
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ppat-1003705-g003: T cell reactivity to in vitro-mannosylated rApa C-terminal.(A) Pooled splenocytes from BCG infected mice (n = 4) were stimulated in vitro with nApa, rApa or 32 individual non-glycosylated synthetic Apa peptides in ELISPOT assay. IFN-γ SFU/106 cells at 12 and 32 wk time points are shown. (B–C) Synthetic C-terminal glycopeptide (Gp281-325)- or control non-glycopeptide (NGp281-325)-specific T cell response (indicated inside dotted box) in mice. Stimulation with proteins (nApa or rApa) or non-glycopeptides (p271-288 or p301-320) was also included as controls. (B) Ag- or peptide-specific IFN-γ, IL-17 or IL-4 SFU/106 pooled lung or spleen cells of BCG or Mtb infected mice at 32 and 26 wks, respectively. (C) The proportions of peptide-specific 1+, 2+ and 3+ cytokine producing T cell subsets constituting total cytokine positive (+) CD4+ T cells in the spleen of BCG infected mice at 32 wks, expressed as % of CD4+ T cells. Data in A–C are means ± s.e.m. *Significant using 1-way ANOVA followed by Bonferroni's test.

Mentions: Thirty-two non-modified, synthetic overlapping peptides of Apa were screened for their capacity to induce IFN-γ response in mice at 12 and 32 wks post BCG infection. Only peptide p271-288 induced a positive response which only occurred 32 wks after infection (Figure 3A). These analyses support the indication that heightened T cell responses to nApa are mainly due to glycosylation. We synthesized rApa C-terminal peptide (residues 281–325; 45-mer) with di-mannosyl-threonine residue at position 316 (Fig. S3A), akin to that found in nApa, to confirm the role of Apa glycosylation in T cell antigenicity.


O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Nandakumar S, Kannanganat S, Dobos KM, Lucas M, Spencer JS, Fang S, McDonald MA, Pohl J, Birkness K, Chamcha V, Ramirez MV, Plikaytis BB, Posey JE, Amara RR, Sable SB - PLoS Pathog. (2013)

T cell reactivity to in vitro-mannosylated rApa C-terminal.(A) Pooled splenocytes from BCG infected mice (n = 4) were stimulated in vitro with nApa, rApa or 32 individual non-glycosylated synthetic Apa peptides in ELISPOT assay. IFN-γ SFU/106 cells at 12 and 32 wk time points are shown. (B–C) Synthetic C-terminal glycopeptide (Gp281-325)- or control non-glycopeptide (NGp281-325)-specific T cell response (indicated inside dotted box) in mice. Stimulation with proteins (nApa or rApa) or non-glycopeptides (p271-288 or p301-320) was also included as controls. (B) Ag- or peptide-specific IFN-γ, IL-17 or IL-4 SFU/106 pooled lung or spleen cells of BCG or Mtb infected mice at 32 and 26 wks, respectively. (C) The proportions of peptide-specific 1+, 2+ and 3+ cytokine producing T cell subsets constituting total cytokine positive (+) CD4+ T cells in the spleen of BCG infected mice at 32 wks, expressed as % of CD4+ T cells. Data in A–C are means ± s.e.m. *Significant using 1-way ANOVA followed by Bonferroni's test.
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Related In: Results  -  Collection

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ppat-1003705-g003: T cell reactivity to in vitro-mannosylated rApa C-terminal.(A) Pooled splenocytes from BCG infected mice (n = 4) were stimulated in vitro with nApa, rApa or 32 individual non-glycosylated synthetic Apa peptides in ELISPOT assay. IFN-γ SFU/106 cells at 12 and 32 wk time points are shown. (B–C) Synthetic C-terminal glycopeptide (Gp281-325)- or control non-glycopeptide (NGp281-325)-specific T cell response (indicated inside dotted box) in mice. Stimulation with proteins (nApa or rApa) or non-glycopeptides (p271-288 or p301-320) was also included as controls. (B) Ag- or peptide-specific IFN-γ, IL-17 or IL-4 SFU/106 pooled lung or spleen cells of BCG or Mtb infected mice at 32 and 26 wks, respectively. (C) The proportions of peptide-specific 1+, 2+ and 3+ cytokine producing T cell subsets constituting total cytokine positive (+) CD4+ T cells in the spleen of BCG infected mice at 32 wks, expressed as % of CD4+ T cells. Data in A–C are means ± s.e.m. *Significant using 1-way ANOVA followed by Bonferroni's test.
Mentions: Thirty-two non-modified, synthetic overlapping peptides of Apa were screened for their capacity to induce IFN-γ response in mice at 12 and 32 wks post BCG infection. Only peptide p271-288 induced a positive response which only occurred 32 wks after infection (Figure 3A). These analyses support the indication that heightened T cell responses to nApa are mainly due to glycosylation. We synthesized rApa C-terminal peptide (residues 281–325; 45-mer) with di-mannosyl-threonine residue at position 316 (Fig. S3A), akin to that found in nApa, to confirm the role of Apa glycosylation in T cell antigenicity.

Bottom Line: Glycosylation is the most abundant post-translational polypeptide chain modification in nature.Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood.These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Tuberculosis Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.

ABSTRACT
Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

Show MeSH
Related in: MedlinePlus