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-7, TNF-α, 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.

Evolution of Drug-Resistant Mycobacterium tuberculosis Strains and Their Adaptation to the Human Lung Environment

“…To date, multiple mycobacterial mannoproteins have been identified, with potential roles in virulence, cell invasion, evasion of host defense, and host immunomodulation, reviewed in Mehaffy et al (2019) , Deng et al (2020) , and Tonini et al (2020) . Many of these antigenic glycoproteins contain acyl groups and are defined as lipoglycoproteins ( Becker and Sander, 2016 ).…”

In-vivo expressed Mycobacterium tuberculosis antigens recognised in three mouse strains after infection and BCG vaccination

“…In addition, Rv0470c prevents phagosome maturation in human monocyte-derived macrophages, while in mice, its deletion alters the persistence and pathology of Mtb at late stages of infection 43 , 45 . Of note, immunisation with BCG_0470 increased the delayed-type hypersensitivity as well as Th1 responses in BCG primed BALB/c mice 46 . All these features, supported by our data in mice and humans 9 , 10 , point to Rv0470c as an attractive BCG booster vaccine candidate when delivered with a properly selected adjuvant 47 .…”

Metabolic Labeling of Bacterial Glycans

“…It should be underlined here that such kind of mannosylation is clearly different from that found in the mannosylated glycans of human cells. [56][57][58] Furthermore, some unusual monosaccharide components such as pseudaminic acid (Pse), legionaminic acid (Leg), bacillosamine (Bac) derivatives, DATDH (2,4-diacetimido-2,4,6-trideoxyhexose), or N-acetyl-D-fucosamine (FucNAc) have been identified in different bacterial species (Fig. 4).…”