10 research outputs found
Structure-Activity Relationship of Gelatinase Biosynthesis-Activating Pheromone of Enterococcus faecalisâ–¿
The expression of pathogenicity-related extracellular proteases, namely, gelatinase and serine protease, in Enterococcus faecalis is positively regulated by a quorum-sensing system mediated by an autoinducing peptide called gelatinase biosynthesis-activating pheromone (GBAP). GBAP is an 11-amino-acid-residue cyclic peptide containing a lactone linkage. To study the structure-activity relationship of GBAP, we synthesized a series of GBAP analogues and evaluated their activities by a gelatinase-inducing assay and newly developed receptor-binding assays in which fluorescence-labeled peptides bound onto the FsrC-overexpressing Lactococcus lactis cell surface were observed by fluorescent microscopy and quantified by using a fluorophotometer. Alanine-scanning analysis of GBAP showed that the entire ring region was involved in the GBAP agonist activity, while side chains of the tail region were not strictly recognized. The alanine substitution of Phe7 or Trp10 almost abolished their receptor-binding abilities and GBAP agonist activities, suggesting that these two aromatic side chains are strongly involved in receptor interaction and activation. Furthermore, the Trp10 substitution with natural and unnatural aromatic amino acids, except pentafluorophenylalanine, caused no loss of agonist activity. This suggested the importance of a negative electrostatic potential created by an π-electron cloud on the aromatic ring surface. Structural analysis of GBAP with nuclear magnetic resonance spectroscopy revealed that the ring region adopted a hairpin-like fold and was tightly packed into a compact form. The side chain of Trp10 was partially buried in the core structure, contributing to the stabilization of the compact form, while that of Phe7 was extended from the core structure into the solvent and was probably directly involved in receptor binding
Expression, purification and activities of the entire family of intact membrane sensor kinases from Enterococcus faecalis
Two-component signal transduction systems are the main mechanism by which bacteria sense and respond to their environment, and their membrane-located histidine protein kinases generally constitute the sensory components of these systems. Relatively little is known about their fundamental mechanisms and precise nature of the molecular signals sensed, because of the technical challenges of producing sufficient quantities of these hydrophobic membrane proteins. This study evaluated the heterologous production, purification and activities of the 16 intact membrane sensor kinases of Enterococcus faecalis. Following the cloning of the genes into expression plasmid pTTQ18His, all but one kinase was expressed successfully in Escherichia coli inner membranes. Purification of the hexa-histidine 'tagged' recombinant proteins was achieved for 13, and all but one were verified as intact. Thirteen intact kinases possessed autophosphorylation activity with no added signal when assayed in membrane vesicles or as purified proteins. Signal testing of two functionally-characterized kinases, FsrC and VicK, was successful examplifying the potential use of in vitro activity assays of intact proteins for systematic signal identification. Intact FsrC exhibited an approximately 10-fold increase in activity in response to a two-fold molar excess of synthetic GBAP pheromone, whilst glutathione, and possibly redox potential, were identified for the first time as direct modulators of VicK activity in vitro. The impact of DTT on VicK phosphorylation resulted in increased levels of phosphorylated VicR, the downstream response regulator, thereby confirming the potential of this in vitro approach for investigations of modulator effects on the entire signal transduction process of two-component systems
Anti-HIV siamycin I directly inhibits autophosphorylation activity of the bacterial FsrC quorum sensor and other ATP-dependent enzyme activities
Siamycin I disrupts growth and quorum sensing in Enterococcus faecalis. Using purified intact protein, we demonstrate here that quorum membrane sensor kinase FsrC is a direct target of siamycin I, reducing pheromone-stimulated autophosphorylation activity by up to 91%. Inhibition was non-competitive with ATP as substrate. Other ATP-binding enzymes were also inhibited, including nine other membrane sensor kinases of E. faecalis, Rhodobacter sphaeroides PrrB, porcine Na +-dependent ATPase and the catalytic subunit of bovine protein kinase A, but not bacterial β-galactosidase, confirming targeted inhibition of a wide range of ATP dependent reactions, and elucidating a likely mechanism underlying the lethality of the inhibitor
Development of a Peptide Antagonist against <i>fsr</i> Quorum Sensing of <i>Enterococcus faecalis</i>
<i>Enterococcus faecalis fsr</i> quorum sensing
(QS)
involves an 11-residue cyclic peptide named gelatinase biosynthesis-activating
pheromone (GBAP) that autoinduces two pathogenicity-related extracellular
proteases in a cell density-dependent fashion. To identify anti-pathogenic
agents that target <i>fsr</i> QS signaling, peptide antagonists
of GBAP were created by our unique drug design approach based on reverse
alanine scanning. First of all, a receptor-binding scaffold (RBS),
[Ala<sup>4,5,6,8,9,11</sup>]ÂZ-GBAP, was created, in which all amino
acids within the ring region of GBAP, except for two essential aromatic
residues, were substituted to alanine. Next, the substituted alanine
residues were changed back to the original amino acid one by one,
permitting selection of those peptide combinations exhibiting increased
antagonist activity. After three cycles of this reverse alanine scan,
[Ala<sup>5,9,11</sup>]ÂZ-GBAP was obtained as a maximally reverted
peptide (MRP) holding the strongest antagonist activity. Then, the
fifth residue in MRP, which is one of the critical residues to determine
agonist/antagonist activity, was further modified by substituting
with different types of amino acids including unnatural amino acids.
As a result, [TyrÂ(Bzl)<sup>5</sup>, Ala<sup>9,11</sup>]ÂZ-GBAP, named
ZBzl-YAA5911, showed the strongest antagonist activity [IC<sub>50</sub> = 26.2 nM and <i>K</i><sub>d</sub> against GBAP receptor
(FsrC) = 39.4 nM]. <i>In vivo</i> efficacy of this peptide
was assessed with an aphakic rabbit endophthalmitis model. ZBzl-YAA5911
suppressed the translocation of <i>E. faecalis</i> from
the aqueous humor into the vitreous cavity by more than 1 order of
magnitude and significantly reduced retinal damage. We propose that
ZBzl-YAA5911 or its derivatives would be useful as anti-infective
agents to attenuate virulence expression in this opportunistic pathogen