Methylation, crystallization and SAD phasing of the Csu pilus CsuC-CsuE chaperone-adhesin subunit pre-assembly...

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Acinetobacter baumannii is a challenging Gram-negative pathogen known for forming biofilms on medical devices and hospital surfaces.
• The Csu pili, assembled via the chaperone-usher pathway, are key to the bacterium's attachment to abiotic surfaces.

Purpose of the Study:

• To determine the crystal structure of CsuE, the pilus tip adhesin of Acinetobacter baumannii.
• To elucidate the mechanism of bacterial attachment to abiotic surfaces.

Main Methods:

• Purification of the CsuC-CsuE chaperone-subunit pre-assembly complex from Escherichia coli.
• Overcoming crystallization challenges through lysine methylation.
• X-ray crystallography of the CsuC-CsuE complex, obtaining data to 2.31 Å resolution.
• Phase determination using single anomalous diffraction with a selenomethionine derivative.

Main Results:

• The CsuC-CsuE complex was successfully crystallized using the hanging-drop vapour-diffusion method.
• Triclinic space group P1 crystals were obtained with specific unit-cell parameters.
• The crystal structure provided insights into the CsuE adhesin's role in pilus assembly and bacterial adhesion.

Conclusions:

• The determined crystal structure of the CsuC-CsuE complex provides a structural basis for understanding Csu pilus assembly in Acinetobacter baumannii.
• This structural information is vital for developing novel strategies to combat biofilm formation and infections caused by this difficult-to-treat pathogen.