ABSTRACT
Virulence factor regulator (Vfr) enhances Pseudomonas aeruginosa pathogenicity through its role as a global transcriptional regulator. The crystal structure of Vfr shows that it is a winged-helix DNA-binding protein like its homologue cyclic AMP receptor protein (CRP). In addition to an expected primary cyclic AMP-binding site, a second ligand-binding site is nestled between the N-terminal domain and the C-terminal helix-turn-helix domain. Unlike CRP, Vfr is a symmetric dimer in the absence of DNA. Removal of seven disordered N-terminal residues of Vfr prevents the growth of P. aeruginosa.
Subject(s)
Bacterial Proteins/metabolism , Cyclic AMP Receptor Protein/metabolism , Gene Expression Regulation, Bacterial/physiology , Pseudomonas aeruginosa/metabolism , Virulence Factors/metabolism , Bacterial Proteins/genetics , Cloning, Molecular , Crystallization , Cyclic AMP Receptor Protein/genetics , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Pseudomonas aeruginosa/genetics , Virulence Factors/geneticsABSTRACT
The transcription factor QscR is a regulator of quorum sensing in Pseudomonas aeruginosa and plays a role in controlling virulence in this prevalent opportunistic pathogen. This study outlines the discovery of a set of synthetic N-acylated homoserine lactones that are capable of either activating or strongly inhibiting QscR in a cell-based reporter gene assay. We demonstrate that the synthetic antagonists inhibit ligand-dependent QscR binding to DNA. Several of these ligands can selectively modulate QscR instead of LasR, or modulate the activity of both receptors, and represent new chemical tools to study the hierarchy of quorum-sensing signaling in P. aeruginosa.
Subject(s)
Acyl-Butyrolactones/chemical synthesis , Acyl-Butyrolactones/pharmacology , Bacterial Proteins/agonists , Bacterial Proteins/antagonists & inhibitors , Drug Design , Pseudomonas aeruginosa/drug effects , Repressor Proteins/agonists , Repressor Proteins/antagonists & inhibitors , Acyl-Butyrolactones/chemistry , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , Dose-Response Relationship, Drug , Ligands , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Pseudomonas aeruginosa/metabolism , Repressor Proteins/metabolism , Signal Transduction/drug effects , Virulence Factors/agonists , Virulence Factors/antagonists & inhibitors , Virulence Factors/metabolismABSTRACT
PilT is a hexameric ATPase required for bacterial type IV pilus retraction and surface motility. Crystal structures of ADP- and ATP-bound Aquifex aeolicus PilT at 2.8 and 3.2 A resolution show N-terminal PAS-like and C-terminal RecA-like ATPase domains followed by a set of short C-terminal helices. The hexamer is formed by extensive polar subunit interactions between the ATPase core of one monomer and the N-terminal domain of the next. An additional structure captures a nonsymmetric PilT hexamer in which approach of invariant arginines from two subunits to the bound nucleotide forms an enzymatically competent active site. A panel of pilT mutations highlights the importance of the arginines, the PAS-like domain, the polar subunit interface, and the C-terminal helices for retraction. We present a model for ATP binding leading to dramatic PilT domain motions, engagement of the arginine wire, and subunit communication in this hexameric motor. Our conclusions apply to the entire type II/IV secretion ATPase family.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/physiology , Bacterial Proteins/chemistry , Bacterial Proteins/physiology , Fimbriae, Bacterial/physiology , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/physiology , Movement/physiology , Protein Subunits/chemistry , Protein Subunits/physiology , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Arginine/chemistry , Bacterial Proteins/genetics , Crystallography, X-Ray , Fimbriae, Bacterial/genetics , Molecular Motor Proteins/genetics , Molecular Sequence Data , Protein Structure, Tertiary/genetics , Protein Subunits/geneticsABSTRACT
PilT is a biological motor required for the retraction of bacterial type IV pili. Nesseria gonorrhoeae PilT has been purified and its ultrastructure has been examined by freeze-etch electron microscopy, revealing a 115 A outer diameter, 15-35 A inner diameter ring. Aquifex aeolicus PilT crystals were obtained in a primitive hexagonal space group (unit-cell parameters a = b = 107.3, c = 68.5 A) and diffract to a minimum Bragg spacing of 2.8 A when PilT is co-crystallized with adenine nucleotides. Initial phases to 3.5 A resolution have been determined by multiwavelength anomalous dispersion and density modification. Resulting electron-density maps show a hexameric A. aeolicus PilT ring 105 A wide by 55 A high, with an inner cavity that varies in shape and width from 20 to 40 A over the height of the complex. Both PilT ultrastructures are very similar to type II and type IV secretion ATPases in overall shape, size and assembly.