Potential Plasticity of the Mannoprotein Repertoire Associated to Mycobacterium tuberculosis Virulence Unveiled by Mass Spectrometry-Based Glycoproteomics

Abstract

To date, Mycobacterium tuberculosis (Mtb) remains the world’s greatest infectious killer. The rise of multidrug-resistant strains stresses the need to identify new therapeutic targets to fight the epidemic. We previously demonstrated that bacterial protein-O-mannosylation is crucial for Mtb infectiousness, renewing the interest of the bacterial-secreted mannoproteins as potential drug-targetable virulence factors. The difficulty of inventorying the mannoprotein repertoire expressed by Mtb led us to design a stringent multi-step workflow for the reliable identification of glycosylated peptides by large-scale mass spectrometry-based proteomics. Applied to the differential analyses of glycoproteins secreted by the wild-type Mtb strain—and by its derived mutant invalidated for the protein-O-mannosylating enzyme PMTub—this approach led to the identification of not only most already known mannoproteins, but also of yet-unknown mannosylated proteins. In addition, analysis of the glycoproteome expressed by the isogenic recombinant Mtb strain overexpressing the PMTub gene revealed an unexpected mannosylation of proteins, with predicted or demonstrated functions in Mtb growth and interaction with the host cell. Since in parallel, a transient increased expression of the PMTub gene has been observed in the wild-type bacilli when infecting macrophages, our results strongly suggest that the Mtb mannoproteome may undergo adaptive regulation during infection of the host cells. Overall, our results provide deeper insights into the complexity of the repertoire of mannosylated proteins expressed by Mtb, and open the way to novel opportunities to search for still-unexploited potential therapeutic targets.

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 ).…”

“…Other groups of glycoproteins involved in M.tb –host interactions are the ones belonging to the MCE family, MPT64, or Apa, among others, involved in colonization and invasion of host cells ( Sonawane et al, 2012 ), as well as other proteins such as Rv0227c, HtrA-like serine protease Rv1223, TatA, GlnA1, and the disulfide oxidase DsbA-like enzyme Rv2969c, recently described as mannosylated ( Tonini et al, 2020 ). In addition, GroEL2, a chaperone-like M.tb capsule-associated glycoprotein, is shown to contribute to the suboptimal antigen presentation during mycobacterial infection by modulating macrophage and DC proinflammatory responses ( Georgieva et al, 2018 ), supporting the immunogenic role of M.tb cell envelope-associated glycoproteins during M.tb infection.…”

Disruption of MenT2 toxin impairs the growth of Mycobacterium tuberculosis in guinea pigs

“…tuberculosis strains. We quantified the transcript levels of Erd_0925 (secreted lipoprotein), Erd_0927 (MenA2), Erd_0929 (glycosylated protein) and Erd_0930 (conserved hypothetical protein) in wild-type and ΔmenT2 strains [70, 71]. The relative transcript levels of menT2 -adjacent genes were comparable in both strains ().…”

“…The lack of complete restoration of the growth defect for the ΔmenT2 mutant strain in guinea pigs prompted us to investigate the effect of deletion of MenT2 on the expression of neighbouring genes and on lipid profiles of M. tuberculosis strains. We quantified the transcript levels of Erd_0925 (secreted lipoprotein), Erd_0927 (MenA2), Erd_0929 (glycosylated protein) and Erd_0930 (conserved hypothetical protein) in wild-type and ΔmenT2 strains [70,71]. The relative transcript levels of menT2-adjacent genes were comparable in both strains (Fig.…”

Tween 80 Improves the Acid-Fast Bacilli Quantification in the Magnetic Nanoparticle-Based Colorimetric Biosensing Assay (NCBA)

“…The core principle of NCBA is cell enrichment through GMNP, due to its glycan interaction with the Mtb cell envelope, which is rich in complex carbohydrates, glycoproteins, and lipids [ 23 , 24 ]. These characteristics allow the formation of complexes between GMNP-AFB by Brownian motion, hydrodynamic force, and physicochemical and molecular interactions.…”