Dataset Information

Expression, purification and crystallization of the phosphate-binding PstS protein from Pseudomonas aeruginosa.

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 C222?, 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 P2?2?2?, 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.

SUBMITTER: Neznansky A

PROVIDER: S-EPMC4089529 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Expression, purification and crystallization of the phosphate-binding PstS protein from Pseudomonas aeruginosa.

Neznansky Avi A Opatowsky Yarden Y

Acta crystallographica. Section F, Structural biology communications 20140618 Pt 7

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 biofilm ...[more]

PMID: 25005086

Similar Datasets

Project description:The increasing prevalence of multi-drug-resistant (MDR) strains of Pseudomonas aeruginosa among critically ill humans is of significant concern. In the current study, we show that MDR clinical isolates of P. aeruginosa representing three distinct genotypes that display high virulence against intestinal epithelial cells, form novel appendage-like structures on their cell surfaces. These appendages contain PstS, an extracellular phosphate binding protein. Using anti-PstS antibodies, we determined that the PstS-rich appendages in MDR strains are involved in adherence to and disruption of the integrity of cultured intestinal epithelial cell monolayers. The outer surface-expressed PstS protein was also identified to be present in P. aeruginosa MPAO1, although to a lesser degree, and its role in conferring an adhesive and barrier disruptive phenotype against intestinal epithelial cells was confirmed using an isogenic DeltaPstS mutant. Formation of the PstS rich appendages was induced during phosphate limitation and completely suppressed in phosphate-rich media. Injection of MDR strains directly into the intestinal tract of surgically injured mice, a known model of phosphate limitation, caused high mortality rates (60%-100%). Repletion of intestinal phosphate in this model completely prevented mortality. Finally, significantly less outer surface PstS was observed in the MPAO1 mutant DeltaHxcR thus establishing a role for the alternative type II secretion system Hxc in outer surface PstS expression. Gene expression analysis performed by RT-PCR confirmed this finding and further demonstrated abundant expression of pstS analogous to pa5369, pstS analogous to pa0688/pa14-55410, and hxcX in MDR strains. Taken together, these studies provide evidence that outer surface PstS expression confers a highly virulent phenotype of MDR isolates against the intestinal epithelium that alters their adhesive and barrier disrupting properties against the intestinal epithelium.

Dataset Information

Expression, purification and crystallization of the phosphate-binding PstS protein from Pseudomonas aeruginosa.

Publications

Expression, purification and crystallization of the phosphate-binding PstS protein from Pseudomonas aeruginosa.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets