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In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm and subsequent derepression of AI-2-related genes necessary for quorum development. While LsrK is a critical enzyme within the DPD quorum sensing relay system, kinetic details of this kinase have yet to be reported. A continuous UV-vis spectrophotometric assay was developed that allowed steady-state kinetic analysis of LsrK to be undertaken with the substrates ATP and DPD. The data was most consistent with a rapid equilibrium ordered mechanism with ATP binding first: kcat (7.4 ± 0.6 s(-1)), Km,ATP (150 ± 30 μM) and Km(app),DPD (1.0 ± 0.2 mM). The assay also allowed a DPD substrate profile to be conducted, which provided an unexpected biochemical disconnect between the previous agonist/antagonist cell-based reporter assay and the LsrK assay presented herein. Together these findings raise the importance of LsrK and lay the foundation not only for further understanding of this enzyme and its critical biological role but also for the rational design of regulatory molecules targeting AI-2 quorum sensing in pathogenic bacteria.

Alkynyl- and azido-tagged 3-oxo-C(12)-acylhomoserine lactone probes have been synthesized to examine their potential utility as probes for discovering the mammalian protein target of the Pseudomonas aeruginosa autoinducer, 3-oxo-C(12)-acylhomoserine lactone. Although such substitutions are commonly believed to be quite conservative, from these studies, we have uncovered a drastic difference in activity between the alkynyl- and azido-modified compounds, and provide an example where such structural modification has proved to be much less than conservative., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Quorum sensing (QS) has traditionally referred to a mechanism of communication within a species of bacteria. However, emerging research implicates QS in interspecies communication and competition, and such systems have been proposed in a wide variety of bacteria. The AI-2-based QS system represents the most studied of these proposed interspecies systems, and has been proposed to regulate diverse functions such as bioluminescence, expression of virulence factors, and biofilm formation. As such, the development of modulatory compounds, both agonists and antagonists, is of great interest for the treatment of bacterial infections and the study of unknown AI-2-based QS systems. Toward this end, we have designed and synthesized a panel of 4,5-dihydroxy-2,3-pentanedione/AI-2 analogues and evaluated their effects on the AI-2 QS of various bacteria. The panel of compounds exhibited differential effects in the bacterial cell lines examined, providing a platform for the development of broad-spectrum modulators of AI-2-based QS.

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa. Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL). [ABSTRACT FROM AUTHOR]

The control of innate immune responses through activation of the nuclear transcription factor NF-B is essential for the elimination of invading microbial pathogens. We showed that the bacterial N-(3-oxo-dodecanoyl) homoserine lactone (C12) selectively impairs the regulation of NF-κB functions in activated mammalian cells. The consequence is specific repression of stimulus-mediated induction of NF-κB-responsive genes encoding inflammatory cytokines and other immune regulators. These findings uncover a strategy by which C12-producing opportunistic pathogens, such as Pseudomonas aeruginosa, attenuate the innate immune system to establish and maintain local persistent infection in humans, for example, in cystic fibrosis patients. [ABSTRACT FROM AUTHOR]

Abstract: The Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, uses acyl-homoserine lactone-based quorum sensing systems to control its pathogenicity. One of its quorum sensing factors, N-3-oxo-dodecanoyl-homoserine lactone, has been shown not only to mediate bacterial quorum sensing but also to exert cytotoxic effects on mammalian cells. The monoclonal antibody RS2-1G9 generated against a 3-oxo-dodecanoyl-homoserine lactone analogue hapten was able to protect murine bone marrow-derived macrophages from the cytotoxic effects and also prevented the activation of the mitogen-activated protein kinase p38. These data demonstrate that an immunopharmacotherapeutic approach to combat P. aeruginosa infections might be a viable therapeutic option as the monoclonal antibody RS2-1G9 can readily sequester bacterial N-3-oxo-dodecanoyl-homoserine lactone molecules, thus interfering with their biological effects in prokaryotic and eukaryotic systems. [Copyright &y& Elsevier]

Abstract: A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody-combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short, narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody-combining site with a closed binding pocket. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will aid further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing. [Copyright &y& Elsevier]

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Here we have shown that p38 and eIF2α phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa. [ABSTRACT FROM AUTHOR]

The article discusses the interference of N-Acyl L-homoserine lactone-based (AHL) quorum sensing signaling as a new approach for anti-infective therapy in the U.S. The interference focused on synthesizing a set of molecules highly congruent in structure to AHL. This paper investigates the stability of several AHL under physiological conditions.