Tyrosine-modifying glycosylation by Yersinia effectors

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• Bacterial glycosyltransferases are key virulence factors that modify host proteins.
• Mono-O-glycosylation by toxins interferes with host cell functions.
• Yersinia species possess glycosyltransferases with high sequence identity.

Purpose of the Study:

• To characterize two Yersinia glycosyltransferases, YeGT and YkGT.
• To investigate their substrate specificity and structural properties.
• To understand their role in host cell modification and virulence.

Main Methods:

• Sequence analysis and comparison of YeGT and YkGT.
• Enzymatic assays to determine substrate specificity (Rho family proteins).
• Site-directed mutagenesis to identify key residues (e.g., residue 177).
• Crystal structure determination of a truncated YeGT (sYeGT).
• Functional assays using anthrax toxin for protein translocation and cell imaging.

Main Results:

• YeGT and YkGT glycosylate Rho family proteins at tyrosine residues (Tyr-34 in RhoA) with GlcNAc.
• YeGT targets RhoA, B, and C, while YkGT has a broader spectrum, including Rac and Cdc42.
• Residue 177 is crucial for YkGT's broader substrate specificity.
• The crystal structure of sYeGT places it in the GT-A family, similar to toxin glycosyltransferases.
• The N-terminal 16 residues are essential for translocation into host cells.
• Expression of YeGT/YkGT induces host cell morphological changes and actin cytoskeleton alterations.

Conclusions:

• YeGT and YkGT are bacterial effector proteins belonging to the tyrosine glycosylating bacterial glycosyltransferases family.
• Their glycosylation activity and translocation mechanisms contribute to Yersinia virulence.
• Structural and functional similarities to known toxins highlight convergent evolution in bacterial pathogenesis.