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Published on: June 28, 2013
Expression, purification and crystallization of the phosphate-binding PstS protein from Pseudomonas aeruginosa
Avi Neznansky1, Yarden Opatowsky1
1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
Insights
Pseudomonas aeruginosa PstS protein is crucial for biofilm formation and virulence. Researchers determined its crystal structure to understand its dual role in phosphate transport and infection, aiding in developing new treatments.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Pseudomonas aeruginosa (PA) infections are a significant threat, causing fatal lung infections, sepsis, and nosocomial infections.
- PA biofilms are critical for virulence, evading host defenses and antibiotics.
- PstS, a component of the PA phosphate transport system, is vital for biofilm establishment and can be secreted to enhance virulence in drug-resistant strains.
Purpose of the Study:
- To elucidate the dual function of PstS in biofilm formation and phosphate transport.
- To determine the crystal structure of PA PstS.
Main Methods:
- Overexpression and purification of PA PstS in Escherichia coli in the presence of phosphate.
- Crystallization using the vapor-diffusion method.
- X-ray diffraction data collection for two crystal forms.
Main Results:
- Two distinct crystal forms of PA PstS were obtained and characterized.
- Crystal form 1: space group C222₁, dimer in asymmetric unit, 41% solvent content.
- Crystal form 2: space group P2₁2₁2₁, tetramer in asymmetric unit, 42.65% solvent content, exhibiting pseudo-translational symmetry consistent with a filamentous arrangement.
Conclusions:
- The crystal structures provide insights into the molecular mechanisms underlying PstS function in PA.
- Understanding PstS structure and its role in virulence is crucial for developing targeted therapies against PA infections.
- The identified crystal packing may reflect PstS assembly in vivo, contributing to bacterial adhesion and biofilm formation.
Abstract:
Pseudomonas aeruginosa (PA) infections pose a serious threat to human health. PA is a leading cause of fatal lung infections in cystic fibrosis and immune-suppressed patients, of sepsis in burn victims and of nosocomial infections. An important element in PA virulence is its ability to establish biofilms that evade suppression by the host's immune system and antibiotics. PstS, a periplasmic subunit of the Pst phosphate-transport system of PA, plays a critical role in the establishment of biofilms. In some drug-resistant PA strains, PstS is secreted in large quantities from the bacteria, where it participates in the assembly of adhesion fibres that enhance bacterial virulence. In order to understand the dual function of PstS in biofilm formation and phosphate transport, the crystal structure of PA PstS was determined. Here, the overexpression in Escherichia coli and purification of PA PstS in the presence of phosphate are described. Two crystal forms were obtained using the vapour-diffusion method at 20°C and X-ray diffraction data were collected. The first crystal form belonged to the centred orthorhombic space group C222₁, with unit-cell parameters a=67.5, b=151.3, c=108.9 Å. Assuming the presence of a dimer in the asymmetric unit gives a crystal volume per protein weight (VM) of 2.09 Å3 Da(-1) and a solvent content of 41%. The second crystal form belonged to the primitive orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=35.4, b=148.3, c=216.7 Å. Assuming the presence of a tetramer in the asymmetric unit gives a crystal volume per protein weight (VM) of 2.14 Å3 Da(-1) and a solvent content of 42.65%. A pseudo-translational symmetry is present in the P212121 crystal form which is consistent with a filamentous arrangement of PstS in the crystal lattice.
