N -linked protein glycosylation in Nanobdellati (formerly DPANN) archaea and their hosts.

Members of the kingdom Nanobdellati , previously known as DPANN archaea, are characterized by ultrasmall cell sizes and reduced genomes. They primarily thrive through ectosymbiotic interactions with specific hosts in diverse environments. Recent successful cultivations have emphasized the importance of adhesion to host cells for understanding the ecophysiology of Nanobdellati . Cell adhesion is often mediated by cell surface carbohydrates, and in archaea, this may be facilitated by the glycosylated S-layer protein that typically coats their cell surface. In this study, we conducted glycoproteomic analyses on two co-cultures of Nanobdellati with their host archaea, as well as on pure cultures of both host and non-host archaea. Nanobdellati exhibited various glycoproteins, including archaellins and hypothetical proteins, with glycans that were structurally distinct from those of their hosts. This indicated that Nanobdellati autonomously synthesize their glycans for protein modifications probably using host-derived substrates, despite the high energy cost. Glycan modifications on Nanobdellati proteins consistently occurred on asparagine residues within the N-X-S/T sequon, consistent with patterns observed across archaea, bacteria, and eukaryotes. In both host and non-host archaea, S-layer proteins were commonly modified with hexose, N -acetylhexosamine, and sulfonated deoxyhexose. However, the N -glycan structures of host archaea, characterized by distinct sugars such as deoxyhexose, nonulosonate sugar, and pentose at the nonreducing ends, were implicated in enabling Nanobdellati to differentiate between host and non-host cells. Interestingly, the specific sugar, xylose, was eliminated from the N -glycan in a host archaeon when co-cultured with Nanobdella . These findings enhance our understanding of the role of protein glycosylation in archaeal interactions.IMPORTANCE Nanobdellati archaea, formerly known as DPANN, are phylogenetically diverse, widely distributed, and obligately ectosymbiotic. The molecular mechanisms by which Nanobdellati recognize and adhere to their specific hosts remain largely unexplored. Protein glycosylation, a fundamental biological mechanism observed across all domains of life, is often crucial for various cell-cell interactions. This study provides the first insights into the glycoproteome of Nanobdellati and their host and non-host archaea. We discovered that Nanobdellati autonomously synthesize glycans for protein modifications, probably utilizing substrates derived from their hosts. Additionally, we identified distinctive glycosylation patterns that suggest mechanisms through which Nanobdellati differentiate between host and non-host cells. This research significantly advances our understanding of the molecular basis of microbial interactions in extreme environments.