Zheng, X. S. | Search bioRxiv

bioRxiv2026

Identification of novel cellular intermediates unveils unique enzymes for flagellar glycan biosynthesis in Clostridioides difficile

Glycosylation of bacterial surface proteins, such as flagellin (FliC), is important for their function and is often involved in virulence of pathogens. Glycans can be further modified by so-called post-glycosylation modifications (PGMs) often resulting in exclusive molecular structures. In Clostridioides difficile a unique glycan structure (Type A) decorates FliC (which forms the flagellar filament) that consists of an O-linked N-acetyl-{beta}-D-glucosamine (GlcNAc) modified with an N-methyl-L-threonine via a phosphodiester linkage. This PGM is synthesized by a set of four enzymes encoded in one operon (ftaABCD), but the exact biosynthesis pathway and biosynthetic intermediates remain unknown. In this study, we chemically synthesized two hitherto undescribed biosynthetic intermediates that we predicted based on bioinformatic analyses, CDP-threonine and CDP-N-methylthreonine. We showed that they are involved in the Type A PGM biosynthesis, as evidenced by mass spectrometric analyses of extracts of a set of C. difficile mutant strains. Furthermore, we characterized FtaC to be a SAM-dependent CDP-threonine N-methyltransferase, that installs the methyl group on CDP-threonine prior to transfer of the PGM to GlcNAc-FliC, and we revealed FtaD as the CDP-N-methylthreonine:GlcNAc N-methylthreoninephosphotransferase. Finally, using recombinantly expressed FtaC and FtaD in combination with synthetic CDP-threonine, we reconstituted the biosynthesis pathway of the Type A PGM in vitro. Overall, our results open avenues to explore these unique biosynthesis enzymes in molecular detail to provide new points of entry for the development of biosynthesis inhibitors and tools to study the role of this PGM in virulence and flagellar assembly.

microbiology↗

bioRxiv2025

Intracortical Microstimulation Induces Rapid Microglia Process Convergence

Intracortical microstimulation (ICMS) has demonstrated the potential to restore vision and hearing by stimulating relevant cortical regions in both animals and humans, offering significant clinical promise for sensory restoration. While the neuronal response to ICMS has been extensively studied at the cellular level through electrophysiology and two-photon (2P) imaging, the response of non-neuronal cells, particularly microglia, as well as the effects of ICMS on blood-brain barrier (BBB) integrity remain poorly understood. In this study, we applied ICMS under 2P imaging in dual-reporter mice, with green fluorescent protein labeling microglia and a red fluorescent Ca2+ indicator labeling neurons. We also monitored vascular dye leakage to assess BBB integrity throughout the experiment. Using clinically relevant waveform parameters, we tested a range of current amplitudes. Surprisingly, we found that microglia responded rapidly, within 15 minutes of stimulation, by converging their processes (MPC) on areas of high neural activity. The prevalence of MPC increased with higher current amplitudes, but intriguingly, it did not correlate with the strength of the local electric field. Additionally, vascular dye penetration into brain tissue was higher in stimulated animals than in controls and increased with current amplitude. This study reveals a rapid microglia and BBB response to ICMS that has not been reported previously, underscoring the need for further research to fully characterize the biological response to ICMS and establish improved safety standards.

bioengineering↗