Impairment of lysosomal function by Clostridioides difficile TcdB

Area of Science:

• Cell Biology
• Microbiology
• Toxicology

Background:

• Clostridioides difficile toxin B (TcdB) is a key virulence factor responsible for severe intestinal infections.
• TcdB inhibits Rho GTPases through mono-glucosylation, disrupting cellular functions.
• The toxin enters host cells via endocytosis, with its glucosyltransferase domain (GTD) escaping endosomes.

Purpose of the Study:

• To investigate the cellular uptake, localization, and degradation pathways of TcdB.
• To elucidate the impact of TcdB on the endo-lysosomal trafficking and degradation machinery.
• To differentiate TcdB-induced lysosomal dysfunction from other cytopathogenic effects.

Main Methods:

• Confocal microscopy to track TcdB and lysosomal marker LAMP1.
• Pulse-chase assays to monitor TcdB and GTD degradation kinetics.
• Biochemical assays using DQ-Green BSA to assess lysosomal degradation capacity.
• Analysis of autophagosomal marker LC3B and lysosomal membrane integrity markers (Galectin-8, Galectin-3).

Main Results:

• TcdB colocalizes with lysosomes, confirming its endo-lysosomal pathway.
• Endocytosed TcdB is rapidly degraded, while the released GTD persists longer.
• TcdB significantly impairs lysosomal degradation of cargo and causes lysosomal dysfunction.
• Lysosomal dysfunction is independent of lysosome membrane damage and affects autophagic flux.

Conclusions:

• TcdB actively interferes with the host cell's lysosomal degradation pathway.
• Lysosomal dysfunction is a distinct cytopathogenic mechanism of TcdB, separate from necrotic signaling.
• Understanding TcdB's impact on lysosomes is crucial for developing targeted therapies against C. difficile infections.