Three distinct glycosylation pathways are involved in the decoration of $Lactococcus\ lactis$ cell wall glycopolymers

Ilias Theodorou (1, 2) , Pascal Courtin (3, 4) , Irina Sadovskaya (5, 6, 7, 8) , Simon Palussière (4) , François Fenaille (9, 10, 4, 11, 12) , Jennifer Mahony (13, 1, 2) , Marie-Pierre Chapot-Chartier (3, 4) , Douwe van Sinderen (1, 13)

1 UCC - University College Cork
2 School of Microbiology
3 MICALIS - MICrobiologie de l'ALImentation au Service de la Santé
4 Université Paris-Saclay
5 BioEcoAgro - UMR transfrontalière INRAE 1158
6 BPA - Biochimie des Produits Aquatiques
7 ULCO - Université du Littoral Côte d'Opale
8 BioEcoAgro - Equipe 8 - Food and Digestive Microbial Ecosystems: Interactions - Dynamics - Application(s)
9 MetaboHUB
10 MTS - Médicaments et Technologies pour la Santé
11 SPI - Service de Pharmacologie et Immunoanalyse
12 LI-MS - Laboratoire Innovations en Spectrométrie de Masse pour la santé
13 APC Microbiome Ireland

Extracytoplasmic sugar decoration of glycopolymer components of the bacterial cell wall contributes to their structural diversity. Typically, the molecular mechanism that underpins such a decoration process involves a three-component glycosylation system (TGS) represented by an undecaprenyl-phosphate (Und-P) sugar-activating glycosyltransferase (Und-P GT), a flippase, and a polytopic glycosyltransferase (PolM GT) dedicated to attaching sugar residues to a specific glycopolymer. Here, using bioinformatic analyses, CRISPR-assisted recombineering, structural analysis of cell wall–associated polysaccharides (CWPS) through MALDI-TOF MS and methylation analysis, we report on three such systems in the bacterium Lactococcus lactis. On the basis of sequence similarities, we first identified three gene pairs, csdAB, csdCD, and csdEF, each encoding an Und-P GT and a PolM GT, as potential TGS component candidates. Our experimental results show that csdAB and csdCD are involved in Glc side-chain addition on the CWPS components rhamnan and polysaccharide pellicle (PSP), respectively, whereas csdEF plays a role in galactosylation of lipoteichoic acid (LTA). We also identified a potential flippase encoded in the L. lactis genome (llnz_02975, cflA) and confirmed that it participates in the glycosylation of the three cell wall glycopolymers rhamnan, PSP, and LTA, thus indicating that its function is shared by the three TGSs. Finally, we observed that glucosylation of both rhamnan and PSP can increase resistance to bacteriophage predation and that LTA galactosylation alters L. lactis resistance to bacteriocin.

Mots clés: flippase; glycopolymer; lactic acid bacteria; lipoteichoic acid (LTA); phage receptor; glycopolymers; flippases; cell wall; glycosylation; glycosyltransferase; bacteriophage; glycobiology; genomics; peptidoglycan

Domaines: Biochimie, Biologie Moléculaire; Biochimie [q-bio.BM]

Ilias Theodorou, Pascal Courtin, Irina Sadovskaya, Simon Palussière, François Fenaille, et al.. Three distinct glycosylation pathways are involved in the decoration of $Lactococcus\ lactis$ cell wall glycopolymers. Journal of Biological Chemistry, 2020, 295 (16), pp.5519-5532. ⟨10.1074/jbc.RA119.010844⟩. ⟨hal-02565128⟩