O-glycosylation modification is crucial for the function of T9SS core component protein GldL in Cytophaga hutchinsonii.

Abstract

The type IX secretion system (T9SS) in Cytophaga hutchinsonii drives essential processes such as cellulose degradation and gliding motility. The T9SS core complex consists of GldK, GldL, GldM, and GldN. Among these, the inner membrane protein GldL bridges the cytoplasmic and periplasmic modules and interacts with GldM to transduce proton motive force (PMF) into the mechanical energy required for secretion. In this study, we report the discovery of O-glycosylation on GldL. Mass spectrometry identified three modified residues (Thr-161, Ser-166, and Ser-170) within cytoplasmic α-helix structure, an atypical location compared to conventional glycosylation sites in flexible loops or β-turns. We constructed a Twin-Strep II-tagged GldL variant for purification and verified its O-glycosylation status using lectin blotting, glycosidase digestion, and β-elimination assays. Phenotypic analysis of a triple glycosylation-site mutant (GldL-Strep-3') revealed severe defects in bacterial growth, cellulose degradation, and gliding motility. Further investigation showed that the loss of GldL glycosylation disrupts T9SS-dependent protein secretion, leading to aberrant periplasmic accumulation of substrates. Our work not only provides the first evidence of GldL glycosylation but also establishes its critical role in T9SS function. This finding offers a novel perspective for elucidating cellulose degradation in C. hutchinsonii and the structure-function relationship of the T9SS.