Dataset Information

Identification of Secreted O-Mannosylated Proteins From BCG and Characterization of Immunodominant Antigens BCG_0470 and BCG_0980.

ABSTRACT: Bacterial glycoproteins have been investigated as vaccine candidates as well as diagnostic biomarkers. However, they are poorly understood in Mycobacterium bovis strain bacille Calmette-Guérin (BCG), a non-pathogenic model of Mycobacterium tuberculosis. To understand the roles of secreted O-mannosylated glycoproteins in BCG, we conducted a ConA lectin-affinity chromatography and mass spectra analysis to identify O-mannosylated proteins in BCG culture filtrate. Subsequent screening of antigens was performed using polyclonal antibodies obtained from a BCG-immunized mouse, with 15 endogenous O-mannosylated proteins eventually identified. Of these, BCG_0470 and BCG_0980 (PstS3) were revealed as the immunodominant antigens. To examine the protective effects of the antigens, recombinant antigens proteins were first expressed in Mycobacterium smegmatis and Escherichia coli, with the purified proteins then used to boost BCG primed-mice. Overall, the treated mice showed a greater delayed-type hypersensitivity response in vivo, as well as stronger Th1 responses, including higher level of IFN-?, TNF-?, and specific-IgG. Therefore, mannosylated proteins BCG_0470 and BCG_0980 effectively amplified the immune responses induced by BCG in mice. Together, our results suggest that the oligosaccharide chains containing mannose are the antigenic determinants of glycoproteins, providing key insight for future vaccine optimization and design.

SUBMITTER: Deng G

PROVIDER: S-EPMC7082424 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Identification of Secreted O-Mannosylated Proteins From BCG and Characterization of Immunodominant Antigens BCG_0470 and BCG_0980.

Deng Guoying G Zhang Wenli W Ji Na N Zhai Yunpeng Y Shi Xiaoxia X Liu Xin X Yang Shufeng S

Frontiers in microbiology 20200313

Bacterial glycoproteins have been investigated as vaccine candidates as well as diagnostic biomarkers. However, they are poorly understood in <i>Mycobacterium bovis</i> strain bacille Calmette-Guérin (BCG), a non-pathogenic model of <i>Mycobacterium tuberculosis</i>. To understand the roles of secreted O-mannosylated glycoproteins in BCG, we conducted a ConA lectin-affinity chromatography and mass spectra analysis to identify O-mannosylated proteins in BCG culture filtrate. Subsequent screening ...[more]

PMID: 32231652

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Project description:Leishmaniasis is a neglected tropical disease (NTD) caused by parasites belonging to the Leishmania genus for which there is no vaccine available for human use. Thus, the aims of this study are to evaluate the immunoprotective effect of a first-generation vaccine against L. amazonensis and to identify its immunodominant antigens. BALB/c mice were inoculated with phosphate buffer sodium (PBS), total L. amazonensis antigens (TLAs), or TLA with Poly (I:C) and Montanide ISA 763. The humoral and cellular immune response was evaluated before infection. IgG, IgG1, and IgG2a were measured on serum, and IFN-γ, IL-4, and IL-10 cytokines as well as cell proliferation were measured on a splenocyte culture from vaccinated mice. Immunized mice were challenged with 104 infective parasites of L. amazonensis on the footpad. After infection, the protection provided by the vaccine was analyzed by measuring lesion size, splenic index, and parasite load on the footpad and spleen. To identify immunodominant antigens, total proteins of L. amazonensis were separated on 2D electrophoresis gel and transferred to a membrane that was incubated with serum from immunoprotected mice. The antigens recognized by the serum were analyzed through a mass spectrometric assay (LC-MS/MS-IT-TOF) to identify their protein sequence, which was subjected to bioinformatic analysis. The first-generation vaccine induced higher levels of antibodies, cytokines, and cell proliferation than the controls after the second dose. Mice vaccinated with TLA + Poly (I:C) + Montanide ISA 763 showed less footpad swelling, a lower splenic index, and a lower parasite load than the control groups (PBS and TLA). Four immunodominant proteins were identified by mass spectrometry: cytosolic tryparedoxin peroxidase, an uncharacterized protein, a kinetoplast-associated protein-like protein, and a putative heat-shock protein DNAJ. The identified proteins showed high levels of conserved sequence among species belonging to the Leishmania genus and the Trypanosomatidae family. These proteins also proved to be phylogenetically divergent to human and canine proteins. TLA + Poly (I:C) + Montanide ISA 763 could be used as a first-generation vaccine against leishmaniasis. The four proteins identified from the whole-protein vaccine could be good antigen candidates to develop a new-generation vaccine against leishmaniasis.

Dataset Information

Identification of Secreted O-Mannosylated Proteins From BCG and Characterization of Immunodominant Antigens BCG_0470 and BCG_0980.

Publications

Identification of Secreted O-Mannosylated Proteins From BCG and Characterization of Immunodominant Antigens BCG_0470 and BCG_0980.

Similar Datasets

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