Research Article: O-mannosylation of the Mycobacterium tuberculosis Adhesin Apa Is Crucial for T Cell Antigenicity during Infection but Is Expendable for Protection

Date Published: October 10, 2013

Publisher: Public Library of Science

Author(s): Subhadra Nandakumar, Sunil Kannanganat, Karen M. Dobos, Megan Lucas, John S. Spencer, Sunan Fang, Melissa A. McDonald, Jan Pohl, Kristin Birkness, Venkateswarlu Chamcha, Melissa V. Ramirez, Bonnie B. Plikaytis, James E. Posey, Rama Rao Amara, Suraj B. Sable, Padmini Salgame.

http://doi.org/10.1371/journal.ppat.1003705

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.

Partial Text

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), produces an array of protein antigens (Ags), many of which are post-translationally modified [1]–[4], which constitute important determinants of innate and adaptive immune response. As with other pathogens, the post-translational protein modifications influence host interactions. In particular, carbohydrate modification of proteins serves as an efficient ligand for innate C-type lectin receptors (CLRs) present on the antigen presenting cells (APCs). Members of this receptor family play an important role in immune response induction, immune evasion, immune regulation and tolerance [5]. In addition to their role in innate immunity, carbohydrate modifications of protein Ags contribute to B cell epitopes, and it has been recently shown that glycopeptides may constitute a T cell epitope and can induce a strong T cell response [6]. Consequently, many glycoproteins or glyco-conjugates are considered Ags of interest in vaccine development. However, little is known about the role of protein glycosylation in T cell immunity in TB, and a better definition of immune responses to glycoproteins may aid in deciphering their role in protection or pathogenesis of Mtb.

Glycosylation generates an enormous variety of modified protein-derived epitopes. However, the significance of such modified antigenic epitopes in intracellular bacterial pathogens in shaping the T cell repertoire and determining the outcome of an immune response is poorly understood. Here we demonstrate that mannosylation of Mtb Apa (also known as MPT-32 or ModD) is crucial for its T cell antigenicity during mycobacterial infections of humans and mice and provides direct evidence that bacterial mannopeptides are T cell Ags using a synthetic Apa glycopeptide. Previously, glycopeptides containing tumor associated carbohydrates [26], [27], glycopeptides of self-Ags in autoimmune diseases [28], and artificially glycosylated peptides of model Ags [6] have been shown to be recognized by T cells. In antigenic glycopeptides, the peptide backbone usually provides the binding motif that interacts with the MHC molecule, while the glycan provides an important part of the structure (epitope) that is recognized by the T cell receptor [29]. Conversely, pure carbohydrates are usually incapable of MHC binding and T cell stimulation, and due to their haptenic nature requires a carrier. The exception to this immunologic paradigm is zwitterionic polysaccharides which are presented in an MHC class-II restricted manner [30]. Because complex carbohydrates are not removed during processing by DCs, it remains possible that mannosylated peptide epitopes within nApa are recognized in vitro by T cells primed in infected mice and humans [31]. Our synthetic peptide screenings suggest that the recognition of carbohydrate containing epitopes (i.e., glycopeptides) rather than increased Ag uptake, mDCs activation or alternate Ag processing leading to enhanced recognition of other peptide epitopes is the major determining factor for heightened T cell responses to nApa during infection. Mapping of the precise mannosylated epitopes will further help to identify whether mannose binds to the MHC groove along with peptide backbone or interacts strictly with TCR and will provide direct insight into the role of mannosylation in Ag presentation of Apa to T cells.

 

Source:

http://doi.org/10.1371/journal.ppat.1003705

 

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