Triboulet, S. | Search bioRxiv

bioRxiv2019

Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis

Transglutaminase 2 (TG2) is a ubiquitous enzyme with transamidating activity. We report here that the expression and activity of TG2 are enhanced in cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 activity impair bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e. hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis. As a consequence, TG2 activation results in increased protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs.

microbiology↗

bioRxiv2018

Tryptophan fluorescence quenching in β-lactam-interacting proteins is modulated by the structure of intermediates and final products of the acylation reaction

In most bacteria, {beta}-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by L,D-transpeptidases (LDTs), which are promising targets for the development of {beta}-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by {beta}-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model L,D-transpeptidase (Ldtfm) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and {beta}-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldtfm. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the {beta}-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of {beta}-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated {beta}-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the {beta}-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation.

biochemistry↗